CN117979833A

CN117979833A - Purified protein composition and method of production

Info

Publication number: CN117979833A
Application number: CN202280064549.XA
Authority: CN
Inventors: 阿尼凯特·凯乐; 兰詹·帕特奈克
Original assignee: Clara Foods Co
Current assignee: Every Co
Priority date: 2021-07-23
Filing date: 2022-07-22
Publication date: 2024-05-03

Abstract

The present disclosure provides methods for producing recombinant proteins that are substantially free of undesirable byproducts including extracellular polysaccharides and off-flavor components. Purification methods involve anionic resins, cation exchange resins, flocculants, adsorbents and/or treatment with enzymes.

Description

Purified protein composition and method of production

Cross Reference to Related Applications

The application claims the benefit of U.S. provisional application No. 63/225,388 filed on 7-month 23 of 2021 and U.S. provisional application No. 63/225,410 filed on 7-month 23 of 2021, the contents of each of which are incorporated by reference in their entirety.

Background

Desirably, the composition of edible recombinant protein (consumable recombination protein) is free of undesirable manufacturing ingredients, contaminants, and other microbial components and byproducts. In some cases, recombinant microbial cells synthesize measurable amounts of undesired byproducts, and when commercial products are produced, these byproducts must be separated from the desired edible recombinant protein. There remains an unmet need for producing edible recombinant proteins that are substantially free of such undesirable byproducts.

SUMMARY

The present disclosure provides methods for producing an edible recombinant protein that is substantially free of the undesirable byproducts disclosed herein.

One aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises Extracellular Polysaccharide (EPS) and an off-flavor component (off-flavor); processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an anionic resin that is reversibly attached to the recombinant protein and that is substantially unattached to the more than one recombinant cell by-product; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Another aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises one or more cation exchange resins that are reversibly attached to the recombinant protein and that are substantially unattached to the more than one recombinant cell by-product; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

A further aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises a flocculant (flocculant) that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

A further aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an adsorbent that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

In one aspect, the present disclosure provides a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product, wherein the processing step comprises digesting the recombinant protein or an enzyme that digests EPS; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

In another aspect, the present disclosure provides an edible composition obtained by any of the methods disclosed herein.

Additionally, any of the compositions or methods disclosed herein are applicable to any of the compositions or methods disclosed herein. In other words, any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein.

Brief Description of Drawings

The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

Fig. 1-10 are flowcharts illustrating methods for producing the protein products or purified EPS of the present disclosure.

FIG. 11 is a chromatogram showing the separation of recombinant proteins using the typical ion exchange resins listed above. The black line is the absorbance trace at 280nm, indicating protein.

FIG. 12 is a chromatogram of a method of purifying an exemplary protein (illustrative protein) using SP 400 resin. n=3, the dashed line shows the conductivity.

FIG. 13 is a chromatogram of a method of purifying an exemplary protein using a combination of SP 400 and SEPRAGEN S resins (ratio of 2.75:1.25). n=3, the dashed line shows the conductivity.

Fig. 14A is a chromatogram of fractions in a method of purifying an exemplary protein using an anion exchange Capto Q resin. The curve with the first two rounded peaks shows protein elution (280 nm), the corner line shows the elution buffer B used in the method, and the curve shows the use during the method, the curve with the final peak shows conductivity (because the expected conductivity is high during protein elution and very high during CIP elution).

FIG. 14B is a SDS-PAGE gel of the fractions shown in FIG. 14A. Lane 2 is feed, lane 3 is flow through, lane 4 is elution, and lane 5 is clean-in-place (CIP).

Fig. 15 is a graph showing the absorption spectrum from the supernatant studied in example 4. Higher pH showed higher absorbance across the plate, indicating that the protein was more stable at pH6 than pH 4. But at pH 4.0, bentonite BE125 showed the greatest decrease in absorbance between the various adsorbents tested. At 350nm, from top to bottom, the curve is Relizorb SP400,400; no filter aid, pH 4; superimposed EZ DE-Celite 545-no filter aid, pH6; stacked DIAON HPA L-85% deacetylated chitosan; and bentonite BE125.

Fig. 16 is a graph showing EPS analysis of the supernatant produced from example 4.

FIG. 17 is a graph showing analysis of protein of interest from the supernatant of example 4.

Figure 18 compares the control and various reprocessing methods. The curve around the pentagonal central core is the most neutral and most preferred protein product. For taste-related data (pointing to the right of pentagons), the worst expressive person is the control; then heating and vacuum; followed by Ion Exchange (IEX), heating and vacuum; then ethanol; and the best performer is ion exchange. For the data related to appearance (upper), the worst manifestations are heat and vacuum; then a control; followed by IEX, heating and vacuum; then ion exchange; and the best performer is ethanol.

Detailed description of the preferred embodiments

It has been found that when recombinant proteins are produced by fermenting yeast cells such as Pichia (Pichia), recombinant cells likewise produce recombinant cell byproducts. The recombinant cell by-product component may be produced in about equal proportions as the recombinant protein, for example, when the recombinant cell by-product is an Extracellular Polysaccharide (EPS). This results in a lower concentration of recombinant protein in the resulting protein product or edible composition. In some cases, the presence of recombinant cell byproducts in the protein product or edible composition may have a non-preferred taste. Furthermore, the presence of recombinant cell byproducts in the protein product or edible composition will have different properties, such as density, viscosity, gelation, and flavor, relative to a protein product or edible composition that is free of recombinant cell byproducts. Thus, there is a need for a method for processing a composition comprising a recombinant protein and recombinant cell by-products, such as EPS and/or off-flavor components, to separate the recombinant protein from the recombinant cell by-products.

Resin-based purification

One aspect of the present disclosure is a method for preparing a protein product with reduced amounts of recombinant cell byproducts. The method comprises the following steps: obtaining a composition comprising a recombinant protein and recombinant cell by-products; processing the composition under conditions that separate the recombinant protein from the recombinant cell by-products, wherein processing comprises a resin that is reversibly attached to the recombinant protein and that is substantially unattached to the recombinant cell by-products; and collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of recombinant cell by-products. In this method, the recombinant cell by-product is an Extracellular Polysaccharide (EPS) or an off-flavor component.

An exemplary method for producing a composition comprising a recombinant protein and a recombinant cell by-product and separating the recombinant protein from the recombinant cell by-product is shown in fig. 1. The methods of the present aspects can be used to isolate any extracellular product produced during a fermentation process. The recombinant cell byproduct (e.g., EPS or odor component) molecules are not ionic in nature and will flow through the ion exchange column.

In embodiments, the resin is an anion exchanger or the resin is a cation exchange resin. In some cases, the cation exchanger is a strong cation exchange resin or a weak cation exchange resin. In some embodiments, the strong cation exchange resin is a sulfonic acid (sulfostate) resin, or the weak cation exchange resin is a carboxymethyl type resin.

Any commercially available resin capable of binding proteins may be used.

One aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an anionic resin that is reversibly attached to the recombinant protein and that is substantially unattached to the more than one recombinant cell by-product; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

In embodiments, the composition comprising the recombinant protein and more than one recombinant cell by-product is a cell culture medium.

In some embodiments, the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium comprising recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

In various embodiments, the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

In several embodiments, the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

In embodiments, the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

In some embodiments, the anion resin is a strong anion exchange resin or a weak anion exchange resin.

In various embodiments, the anionic resin is one or more of the following: capto Q resin, DEAE type weak anion exchanger, resin with trimethylaminoethyl group, resin with triethylaminoethyl group, resin with quaternary amine group.

In several embodiments, the anionic resin is a component of a chromatographic system.

In embodiments, the chromatography system is operated in batch mode with an axial flow column (axial flow column) or radial flow column (radial flow column) or centrifugal column, or by using a membrane chromatography column.

In some embodiments, the chromatography system operates in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography process (e.g., equilibration, loading, elution, and washing) occur simultaneously.

In various embodiments, the continuous mode includes a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

In several embodiments, a composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove spent biomass (particle biomass) comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

In embodiments, the treatment to remove small non-protein molecules includes the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

In some embodiments, the treatment to remove small non-protein molecules comprises diafiltration buffer.

In various embodiments, the method further comprises a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

In several embodiments, the protein-containing composition having preferred pH and/or ionic conditions is also heat treated and/or dried.

In embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is also heat treated and/or dried.

In some embodiments, the heat treatment separates the recombinant protein from the off-flavor component and applies heat at a temperature and for a duration such that the off-flavor component volatilizes and the gaseous off-flavor component is removable.

In various embodiments, the vacuum is applied simultaneously with the application of heat, and facilitates removal of gaseous off-flavor components.

In several embodiments, the off-flavor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

In embodiments, the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

In some embodiments, the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃, during the heat treatment.

In various embodiments, the method includes agitation during the heat treatment.

In several embodiments, the heat treatment and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

In embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step, e.g., comprising the addition of hydrogen peroxide.

In some embodiments, the ratio of recombinant cell by-products to recombinant proteins in a composition comprising recombinant protein and more than one recombinant cell by-products is about 1:3 to about 3:1.

In various embodiments, the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to a composition comprising the recombinant protein and more than one recombinant cell by-product.

In several embodiments, less than about 10% by weight of the protein product constitutes recombinant cell by-products.

In embodiments, less than about 5% by weight of the protein product constitutes recombinant cell by-products.

In some embodiments, less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises the off-flavor component.

In various embodiments, off-flavor components in the protein product are virtually undetectable to standard consumers.

In several embodiments, EPS is generally inseparable from recombinant proteins when size exclusion chromatography is used.

In embodiments, EPS is naturally a component of the cell wall of recombinant cells.

In some embodiments, EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

In various embodiments, the EPS comprises mannose.

In several embodiments, the EPS further comprises N-acetylglucosamine and/or glucose.

In embodiments, EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose, as analyzed by gas chromatography coupled with mass spectrometry.

In embodiments, EPS comprises an α (1, 6) -linked backbone having α (1, 2) -linked branches and/or α (1, 3) -linked branches.

In various embodiments, the EPS is mannan.

In several embodiments, the recombinant cells expressing the recombinant protein and more than one recombinant cell by-product are selected from fungal cells, such as filamentous fungi or yeast, bacterial cells, plant cells, insect cells, or mammalian cells.

In the context of an embodiment of the present invention, the recombinant cell type is selected from the group consisting of a species of a. Sp., (Arxula spp.), (Arxula adeninivorans), kluyveromyces spp.), (Kluyveromyces lactis), kluyveromyces (Komagataella phaffii), pichia (Pichia spp.), (Angustifolia) Pichia angusta (Pichia angusta), pichia pastoris (Pichia pastoris), yeast species (Saccharomyces spp.), (Saccharomyces cerevisiae), schizosaccharomyces (Schizosaccharomyces spp.), (Schizosaccharomyces pombe), yarrowia (Yarrowia spp.), (Yarrowia lipolytica), agaricus (Agrocaricup spp.), (Agaricus bisporus), aspergillus (Aspergillus acutifolia) species (Agaricus bisporus), pachyrhizus (Aspergillus awamori), aspergillus (26), aspergillus (Aspergillus niger) species (Aspergillus kawachii), aspergillus (35), aspergillus kavalis (35), aspergillus kawachii (35), aspergillus kavalis (Aspergillus kavalis, aspergillus kafimbricus (35), aspergillus kava (35), aspergillus kawachii (35), aspergillus kaki (35), aspergillus kawachii (35), aspergillus kaki) and other than (Fusari (35 Mucor (Mucor pusillus), myceliophthora species (Myceliophora spp.), myceliophthora thermophila (Myceliophthora thermophila), neurospora species (Neurospora spp.), neurospora crassa (Neurospora crassa), penicillium species (Penicillium spp.), penicillium sham (Penicillium camemberti), penicillium griseum cinerea (Penicillium canescens), penicillium chrysogenum (Penicillium chrysogenum), penicillium elmersonii (Penicillium (Talaromyces) emersonii), penicillium ropus (Penicillium funiculosum), penicillium purpurogenum (Penicillium purpurogenum), penicillium romyces roqueforti (Penicillium roqueforti), pleurotus species (Pleurotus spp.), pleurotus (Pleurotus ostreatus), pseudomonas species (Pseudomonas spp.), rhizomucor species (Rhizomucor spp.), rhizopus nii (Rhizomucor miehei), rhizopus nikowii (Rhizomucor pusillus), rhizopus species (Rhizopus spp.), rhizopus (74), rhizopus Trichoderma (Trichoderma reesei), rhizopus (3742, rhizopus viride (3742).

In some embodiments, the fungus is a pichia species.

In various embodiments, the pichia species is saccharomyces falciparum or saccharomyces pastoris (Komagataella pastoris).

In several embodiments, the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

In embodiments, the enzyme is pepsinogen or pepsin.

In some embodiments, the protein is an egg white protein (egg-white protein).

In various embodiments, the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein (ovostatin), a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

In several embodiments, the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an naturally occurring egg white protein in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

In embodiments, the edible composition comprising the protein product comprises a food product, a beverage product, or a dietary supplement.

In some embodiments, the food product includes baked goods (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelets, cream, pasta, noodles, colates, wafer, dough, batter, cookie dough, meat rolls, meatballs, hamburgers, animal feed, fruit, vegetables, tofu (tofu), tofu (bean curd), cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, dessert, marshmallow, soup, condiments, sauce, spice, dairy products, and flavoring (dressings).

In various embodiments, beverage products include soft drinks, flavored waters, juices, sport drinks, energy drinks, smoothies, milkshakes, alcoholic beverages (e.g., wine, sake, beer, spirits), cocktails, liqueurs, carbonated beverages, caffeine-containing beverages, coffee, cocoa, tea, egg and milk drinks, and dairy beverages.

In several embodiments, the dietary (dietary) supplements include multivitamins, whole food supplements, dietary supplements, herbal supplements, protein mixtures, weighting agents, ready-to-drink proteins, protein bars, protein milkshakes, protein powders, protein pellets, protein isolates, energy bars, energy gels, energy chews, energy formulas (energy formulas), endurance formulas (endurance formula), energy supplements, nutritional supplements, sports nutritional supplements, infant formulas (infant formulas) (e.g., powder or liquid), and meal (mean) substitutes.

In embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) One or more cation exchange resins reversibly attached to the recombinant protein and not substantially attached to the EPS, ii) an enzyme that digests the recombinant protein or EPS, iii) an adsorbent reversibly attached to the EPS and not substantially attached to the recombinant protein, and/or iv) a flocculant attached to the EPS and not substantially attached to the recombinant protein.

In another aspect, the present disclosure provides an edible composition obtained by any of the above disclosed methods.

In some embodiments, the one or more cation exchange resins include a strong cation exchange resin, e.g., a sulfopropyl-type resin, a sulfomethyl-type resin, or a sulfonate-type resin, and/or a weak cation exchange resin, e.g., a carboxymethyl-type resin.

In various embodiments, the one or more cation exchange resins include polystyrene divinylbenzene, polymethacrylate, or cellulose or crosslinked dextran or crosslinked agarose or inorganic materials coated with hydrophilic polymers.

In several embodiments, the one or more cation exchange resins have a particle size of about 50 μm and about 200 μm and/or have a protein binding capacity of about 50g protein/L resin to about 100g protein/L resin.

In embodiments, the one or more cation exchange resins include Cytiva Capto S, HP20, relizorb SP400, SEPRAGEN S, SP20, and/or Mitsubishi Relizorb EXE349.

In some embodiments, the processing step includes two cationic resins, and in some cases, the two cationic resins are in a ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.

In various embodiments, the two resins are SP400 and SEPRAGEN S and are in a ratio of about 3:1, e.g., 2.75:1.25.

In several embodiments, the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

In embodiments, the composition comprising the recombinant protein and the more than one recombinant cell by-product has a pH less than the isoelectric point (pI) of the recombinant protein by reducing the pH of the composition comprising the recombinant protein and the more than one recombinant cell by-product.

In some embodiments, the one or more cationic resins are components of a chromatography system, and in some cases, the chromatography system is operated in batch mode with an axial flow column or radial flow column or centrifugal column, or by using a membrane chromatography column.

In various embodiments, the one or more cationic resins are components of a chromatography system, and in some cases, the chromatography system is operated in a continuous mode comprising a plurality of parallel columns, the feed of the columns being switchable such that multiple steps in the chromatography process (e.g., equilibration, loading, elution, and washing) occur simultaneously.

In several embodiments, the continuous mode includes a Simulated Moving Bed (SMB) or ion separator (e.g.,) The system.

In embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

In some embodiments, the treatment to remove small non-protein molecules includes the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

In various embodiments, the treatment to remove small non-protein molecules comprises diafiltration buffer.

In several embodiments, the method further comprises a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

In embodiments, the protein-containing composition having preferred pH and/or ionic conditions is also heat treated and/or dried.

In some embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is also heat treated and/or dried.

In various embodiments, the heat treatment separates the recombinant protein from the off-flavor components, and in some cases, applies heat at a temperature and for a duration such that the off-flavor components volatilize and gaseous off-flavor components are removable.

In several embodiments, a vacuum is applied simultaneously with the application of heat, and the vacuum facilitates removal of gaseous off-flavor components.

In embodiments, the off-flavor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

In some embodiments, the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

In various embodiments, the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃, during the heat treatment.

In several embodiments, the method includes agitation during the heat treatment.

In embodiments, the heat treatment and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

In some embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step, e.g., comprising the addition of hydrogen peroxide.

In various embodiments, the ratio of recombinant cell by-products to recombinant proteins in a composition comprising recombinant protein and more than one recombinant cell by-products is from about 1:3 to about 3:1.

In several embodiments, the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to a composition comprising the recombinant protein and more than one recombinant cell by-product.

In embodiments, less than about 10% by weight of the protein product constitutes recombinant cell by-products.

In some embodiments, less than about 5% by weight of the protein product constitutes recombinant cell by-products.

In various embodiments, less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises the off-flavor component.

In several embodiments, off-flavor components in the protein product are virtually undetectable to standard consumers.

In embodiments, EPS is generally inseparable from recombinant proteins when size exclusion chromatography is used.

In some embodiments, EPS is naturally a component of the cell wall of a recombinant cell.

In various embodiments, EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

In several embodiments, the EPS comprises mannose.

In embodiments, the EPS further comprises N-acetylglucosamine and/or glucose.

In some embodiments, EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose, as analyzed by gas chromatography coupled with mass spectrometry.

In various embodiments, the EPS comprises an α (1, 6) -linked backbone having α (1, 2) -linked branches and/or α (1, 3) -linked branches.

In several embodiments, the EPS is mannan.

In embodiments, the recombinant cell expressing the recombinant protein and more than one recombinant cell by-product is selected from a fungal cell, such as a filamentous fungus or yeast, a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

In some embodiments of the present invention, in some embodiments, the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces cerevisiae, a species of the genus Schizosaccharomyces pombe, a species of the genus Pichia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

In various embodiments, the fungus is a pichia species.

In several embodiments, the pichia species is saccharomyces falciparum or saccharomyces pastoris.

In embodiments, the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

In some embodiments, the enzyme is pepsinogen or pepsin.

In various embodiments, the protein is an egg white protein.

In several embodiments, the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an ovosolid protein, a cysteine protease inhibitor, an avidin, an ovalbumin-related protein X, or an ovalbumin-related protein Y, and any combination thereof.

In embodiments, the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to naturally occurring egg white protein in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

In some embodiments, the edible composition comprising the protein product comprises a food product, a beverage product, or a dietary supplement.

In various embodiments, the food product includes baked goods (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelets, cream, pasta, noodles, colates, wafer, dough, batter, cookie dough, meat rolls, meatballs, hamburgers, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, desserts, marshmallows, soup, condiments, sauce, spices, dairy products, and condiments.

In several embodiments, the beverage products include soft drinks, flavored waters, juices, sport drinks, energy drinks, smoothies, milkshakes, alcoholic beverages (e.g., wine, sake, beer, spirits), cocktails, liqueurs, carbonated beverages, caffeine-containing beverages, coffee, cocoa, tea, egg-milk, and dairy beverages.

In embodiments, the dietary supplements include multivitamins, whole food supplements, dietary supplements, herbal supplements, protein mixtures, weighting agents, ready-to-drink proteins, protein bars, protein milkshakes, protein powders, protein pellets, protein isolates, energy bars, energy gels, energy chews, energy formulas, endurance formulas, energy supplements, nutritional supplements, sports nutritional supplements, infant formulas (e.g., powders or liquids), and dietary substitutes.

In some embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) An anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an enzyme that digests the recombinant protein or EPS, iii) an adsorbent reversibly attached to the EPS and substantially unattached to the recombinant protein, and/or iv) a flocculant attached to the EPS and substantially unattached to the recombinant protein.

In various embodiments, the resin is a component of a chromatography system. In some cases, the chromatography system is operated in batch mode with an axial flow column or radial flow column or centrifugal column, or by using a membrane chromatography column, or the chromatography system is operated in continuous mode comprising multiple parallel columns, the feed to the columns being switchable so that multiple steps in the chromatography process (e.g., equilibration, loading, elution and washing) occur simultaneously. In many cases, the continuous mode includes a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

In embodiments, the composition comprising recombinant protein and recombinant cell byproducts is pre-treated to remove waste biomass comprising recombinant cells and pre-treated to remove small non-protein molecules. In some cases, the treatment to remove small non-protein molecules includes a step of concentrating a composition comprising recombinant protein and recombinant cell by-products. In embodiments, the treatment to remove small non-protein molecules comprises diafiltration buffer.

In some embodiments, the method further comprises a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions. In embodiments, the protein-containing composition having preferred pH and/or ionic conditions is also heat treated and/or dried.

An exemplary chromatogram for purification of recombinant proteins using cation exchange column SP400 is shown in fig. 11. The recombinant protein was bound to the column eluting in elution zones 1 and 2 (starting from 8 minutes and 11 minutes, respectively). More specifically, the fractions in elution zone 2 are recombinant proteins of interest that separate from unbound peaks during loading (about 5 minutes), recombinant proteins of interest that separate from bound product peaks during in-situ washing (CIP; about 16 minutes), and other loosely bound proteins in elution zone 1 (about 8 minutes).

In some embodiments, a variation of the methods shown in fig. 1 or fig. 7 would be to equilibrate the column in elution buffer 1 prior to feed application to elute host cell proteins as well as recombinant cell byproduct impurities, such as EPS or off-flavor components.

In another variation of the method shown in fig. 1 or fig. 7, the concentration step is omitted and the micro-filtered fermentation supernatant is loaded onto a column equilibrated in elution buffer 1; thereby separating recombinant cell byproducts (e.g., EPS or off-flavor components), small molecule impurities, and host cell proteins in a column loading step.

Methods for protein isolation and intracellular protein isolation have been described in the literature, see for example US10,857,483 and US 9,821,249; the contents of each of which are incorporated herein by reference in their entirety.

Purification based on hydrophobic or amphiphilic solvents

One aspect of the present disclosure is a method for preparing a protein product with reduced amounts of recombinant cell byproducts. The method comprises the following steps: obtaining a composition comprising a recombinant protein and recombinant cell by-products; processing the composition under conditions that separate the recombinant protein from the recombinant cell by-products; the isolated recombinant protein is collected, thereby obtaining a protein product having a reduced amount of recombinant cell byproducts. In this method, the recombinant cell by-product is an off-flavor component. In embodiments, the step of processing the composition comprises using a hydrophobic or amphiphilic solvent that separates the recombinant protein from the recombinant cell by-products.

An exemplary method for producing a composition comprising a recombinant protein and a recombinant cell by-product and separating the recombinant protein from the recombinant cell by-product is shown in fig. 6.

In embodiments, the composition comprising the recombinant protein and the recombinant cell by-product is produced by fermentation of a recombinant cell.

In some embodiments, the composition comprising recombinant protein and recombinant cell by-products is pre-treated to remove spent biomass comprising recombinant cells.

In various embodiments, the composition comprising recombinant protein and recombinant cell by-products is pre-treated to remove small non-protein molecules. In some cases, the treatment to remove small non-protein molecules includes diafiltration buffer. The treatment to remove small non-protein molecules may include the step of concentrating the composition comprising recombinant protein and recombinant cell by-products.

In some embodiments, the method further comprises a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions. In some cases, the protein-containing composition having preferred pH and/or ionic conditions is also heat treated and/or dried. The heat treatment and/or drying step may produce a dried protein product having a reduced amount of off-flavor components.

In various embodiments, the protein product having a reduced amount of off-flavor component comprises at least a 50% reduction in the amount of off-flavor component relative to a composition comprising the recombinant protein and recombinant cell by-product. In some cases, the protein product has at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of off-flavor components relative to a composition comprising the recombinant protein and recombinant cell by-products.

In embodiments, less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises the off-flavor component. In some cases, off-flavor components in the protein product are virtually undetectable to standard consumers.

Enzyme-based purification

Exemplary methods for producing a composition comprising a recombinant protein and a recombinant cell by-product and separating the recombinant protein from the recombinant cell by-product are shown in fig. 2 and 8. The methods of the present aspects can be used to isolate any extracellular product produced during a fermentation process.

In embodiments, the enzyme digests the recombinant protein or digests the recombinant cell by-product.

In some embodiments, the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is adjusted to achieve a pH greater than the pI of the recombinant protein.

In various embodiments, the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is about 6.

In several embodiments, the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH less than the isoelectric point (pI) of the recombinant protein.

In embodiments, the pH of a composition comprising a recombinant protein and more than one recombinant cell by-product is achieved by lowering the pH.

In some embodiments, the enzyme that digests the recombinant protein is pepsin or trypsin.

In various embodiments, the digested recombinant protein is permeated through an ultrafiltration system, e.g., having a 10kDa membrane.

In several embodiments, the enzyme that digests EPS is mannanase, cellulase, or glucanase.

In embodiments, the undigested recombinant protein is concentrated by an ultrafiltration system, e.g., having a 5kDa membrane.

In some embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises a chromatography system.

In various embodiments, the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column.

In several embodiments, the chromatography system operates in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography process (e.g., equilibration, loading, elution, and washing) occur simultaneously.

In embodiments, the continuous mode includes a Simulated Moving Bed (SMB) or ion separator (e.g.,) The system.

In some embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

In various embodiments, the treatment to remove small non-protein molecules includes the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

In several embodiments, the treatment to remove small non-protein molecules comprises diafiltration buffer.

In embodiments, the method further comprises a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

In some embodiments, the protein-containing composition having preferred pH and/or ionic conditions is also heat treated and/or dried.

In various embodiments, the composition comprising the recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is also heat treated and/or dried.

In several embodiments, the heat treatment separates the recombinant protein from the off-flavor components, and in some cases, applies heat at a temperature and for a duration such that the off-flavor components volatilize and gaseous off-flavor components are removable.

In embodiments, the vacuum is applied simultaneously with the application of heat, and facilitates removal of gaseous off-flavor components.

In some embodiments, the off-flavor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

In various embodiments, the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

In several embodiments, the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃, during the heat treatment.

In an embodiment, the method comprises stirring during the heat treatment.

In some embodiments, the heat treatment and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

In various embodiments, the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is also subjected to an oxidation step.

In several embodiments, the oxidizing step includes adding hydrogen peroxide.

In embodiments, in a composition comprising a recombinant protein and more than one recombinant cell by-product, the ratio of recombinant cell by-product to recombinant protein is from about 1:3 to about 3:1.

In some embodiments, the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to a composition comprising the recombinant protein and more than one recombinant cell by-product.

In various embodiments, less than about 10% by weight of the protein product constitutes recombinant cell by-products.

In several embodiments, less than about 5% by weight of the protein product constitutes recombinant cell by-products.

In embodiments, less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises the off-flavor component.

In some embodiments, off-flavor components in the protein product are virtually undetectable to standard consumers.

In various embodiments, EPS is generally inseparable from recombinant proteins when size exclusion chromatography is used.

In several embodiments, EPS is naturally a component of the cell wall of a recombinant cell.

In embodiments, EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

In some embodiments, the EPS comprises mannose.

In various embodiments, the EPS further comprises N-acetylglucosamine and/or glucose.

In several embodiments, EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose, as analyzed by gas chromatography coupled with mass spectrometry.

In some embodiments, the EPS is mannan.

In various embodiments, the recombinant cells expressing the recombinant protein and more than one recombinant cell by-product are selected from fungal cells, such as filamentous fungi or yeast, bacterial cells, plant cells, insect cells, or mammalian cells.

In a number of embodiments of the present invention, the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces cerevisiae, a species of the genus Schizosaccharomyces pombe, a species of the genus Pichia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

In embodiments, the fungus is a pichia species.

In some embodiments, the pichia species is saccharomyces falciparum or saccharomyces pastoris.

In various embodiments, the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

In several embodiments, the enzyme is pepsinogen or pepsin.

In embodiments, the protein is an egg white protein.

In some embodiments, the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, an avidin, an ovalbumin-related protein X, or an ovalbumin-related protein Y, and any combination thereof.

In various embodiments, the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an naturally occurring egg white protein in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

In several embodiments, the edible composition comprising the protein product comprises a food product, a beverage product, or a dietary supplement.

In embodiments, the food product includes baked goods (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelets, custards, pasta, noodles, colates, wafer, dough, batter, cookie dough, meat rolls, meatballs, hamburgers, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, milk, foam, meringue, frosting, desserts, marshmallow, soup, condiments, sauce, spices, dairy products, and condiments.

In some embodiments, beverage products include soft drinks, flavored waters, juices, sport drinks, energy drinks, smoothies, milkshakes, alcoholic beverages (e.g., wine, sake, beer, spirits), cocktails, liqueurs, carbonated beverages, caffeine-containing beverages, coffee, cocoa, tea, egg and milk drinks, and dairy beverages.

In various embodiments, the dietary supplements include multivitamins, whole food supplements, dietary supplements, herbal supplements, protein mixtures, weighting agents, ready-to-drink proteins, protein bars, protein milkshakes, protein powders, protein pellets, protein isolates, energy bars, energy gels, energy chews, energy formulas, endurance formulas, energy supplements, nutritional supplements, sports nutritional supplements, infant formulas (e.g., powders or liquids), and meal substitutes.

In several embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of the following: i) A cationic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, iii) a flocculant attached to the EPS and substantially unattached to the recombinant protein, and/or iv) an adsorbent attached to the EPS and substantially unattached to the recombinant protein.

Purification based on heating

One aspect of the present disclosure is a method for preparing a protein product with reduced amounts of recombinant cell byproducts. The method comprises the following steps: obtaining a composition comprising a recombinant protein and recombinant cell by-products; processing the composition under conditions that separate the recombinant protein from the recombinant cell by-products; the isolated recombinant protein is collected, thereby obtaining a protein product having a reduced amount of recombinant cell byproducts. In this method, the recombinant cell by-product is an off-flavor component.

In various embodiments, the step of processing the composition includes using heat to separate the recombinant protein from the recombinant cell byproducts, and, in some cases, applying heat at a temperature and for a duration such that the recombinant cell byproducts volatilize and gaseous recombinant cell byproducts are removable.

In embodiments, the composition may be stirred while heat is applied.

In some embodiments, the vacuum is applied simultaneously with the application of heat. In some cases, the vacuum facilitates removal of gaseous recombinant cell byproducts.

In many cases, the temperature is as high as 80 ℃.

Adsorbent-based purification

An exemplary method for producing a composition comprising a recombinant protein and a recombinant cell by-product and separating the recombinant protein from the recombinant cell by-product is shown in fig. 3 or 9. The methods of the present aspects can be used to isolate any extracellular product produced during a fermentation process.

In various embodiments, the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

In several embodiments, the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

In embodiments, the adsorbent is added to a medium comprising recombinant cells that secrete recombinant proteins and more than one recombinant cell by-product.

In some embodiments, once the adsorbent is attached to one or more components of more than one recombinant cell by-product, the adsorbent is separated from the recombinant protein.

In various embodiments, when the adsorbent is attached to one or more components of more than one recombinant cell by-product, the adsorbent is separated from the recombinant protein with a filter (strainer), a filtration device, and/or by centrifugation.

In several embodiments, the method further comprises replenishing the medium with the adsorbent again.

In embodiments, the adsorbent is provided to the biomass separation feed tank and one or more components of more than one recombinant cell byproduct while the spent biomass comprising the recombinant cells is removed.

In some embodiments, the adsorbent is provided after removal of the spent biomass comprising the recombinant cells.

In several embodiments, the adsorbent comprises a resin and/or a hydrophobic adsorbent comprising, for example, a methacrylate or silica backbone, or is a DEAE-type weak anion exchanger.

In embodiments, the adsorbent is Dow Amberlite SD2, mitsubishi Diaion HP, celite 545, bentonite BE125, DIAION HPA25L, 85% deacetylated chitosan, EZ DE, ultra pure diatomaceous earth, or Relizorb SP.

In some embodiments, the adsorbent is disposed in, for example, a column and operated in batch mode with an axial flow column or radial flow column or centrifugal column, or by using a membrane chromatography column.

In various embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises a chromatography system.

In several embodiments, the chromatography system is operated in batch mode with an axial flow column or radial flow column or centrifugal column, or by using a membrane chromatography column, or the chromatography system is operated in continuous mode comprising a plurality of parallel columns, the feed of the columns being switchable such that multiple steps in the chromatography process (e.g., equilibration, loading, elution and washing) occur simultaneously.

In an embodiment, the method comprises stirring during the heat treatment.

In several embodiments, the oxidizing step includes adding hydrogen peroxide.

In some embodiments, the EPS comprises mannose.

In some embodiments, the EPS is mannan.

In embodiments, the fungus is a pichia species.

In several embodiments, the enzyme is pepsinogen or pepsin.

In embodiments, the protein is an egg white protein.

In several embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of the following: i) A cationic resin that is reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an anionic resin that is reversibly attached to the recombinant protein and substantially unattached to the EPS, iii) an enzyme that digests the recombinant protein or EPS, and/or iv) a flocculant that is attached to the EPS and substantially unattached to the recombinant protein.

In embodiments, the adsorbent is separated from the fermentation medium after the adsorbent is reversibly attached to the recombinant cell byproducts. In some cases, the adsorbent is separated with a filter or other filtration device. In many cases, the fermentation medium is again supplemented with an adsorbent.

Flocculant-based purification

A further aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises a flocculant that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Exemplary methods for producing a composition comprising a recombinant protein and a recombinant cell by-product and separating the recombinant protein from the recombinant cell by-product are shown in fig. 4 or 10. The methods of the present aspects can be used to isolate any extracellular product produced during a fermentation process.

In embodiments, the flocculant is added to a medium comprising recombinant cells that secrete recombinant proteins and more than one recombinant cell by-product.

In some embodiments, once the flocculant is attached to one or more components of more than one recombinant cell byproduct, the flocculant separates from the recombinant protein.

In various embodiments, when the flocculant is attached to one or more components of more than one recombinant cell by-product, the flocculant is separated from the recombinant protein with a filter, filtration apparatus, and/or by centrifugation.

In several embodiments, the method further comprises replenishing the culture medium with a flocculant again.

In embodiments, a flocculant is provided to the biomass separation feed tank and one or more components of more than one recombinant cell byproduct while the spent biomass comprising the recombinant cells is removed.

In some embodiments, the flocculant is provided after removing the spent biomass comprising the recombinant cells.

In several embodiments, the flocculant is an anionic flocculant or a neutral flocculant.

In embodiments, the flocculant is Tramfloc, tramfloc, tramfloc 110, tramfloc or Tramfloc 120, magnafloc 333, magnafloc 355 or Gusmer avergan, dupont Polyox, celite 545, bentonite BE125, DIAION HPA25L, 85% deacetylated chitosan, EZ DE, ultra-pure diatomaceous earth, or Relizorb SP400.

In some embodiments, the flocculant is disposed in a column.

In an embodiment, the method comprises stirring during the heat treatment.

In several embodiments, the oxidizing step includes adding hydrogen peroxide.

In some embodiments, the EPS comprises mannose.

In some embodiments, the EPS is mannan.

In embodiments, the fungus is a pichia species.

In several embodiments, the enzyme is pepsinogen or pepsin.

In embodiments, the protein is an egg white protein.

In several embodiments, processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of the following: i) A cationic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, iii) an enzyme that digests the recombinant protein or EPS, and/or iv) an adsorbent attached to the EPS and substantially unattached to the recombinant protein.

Protein product and edible composition containing protein

Another aspect of the present disclosure is a protein product prepared by any of the methods disclosed herein.

Yet another aspect of the present disclosure is an edible composition comprising any of the protein products disclosed herein.

In one aspect, the present disclosure provides an edible composition for use in a food product as disclosed herein.

In embodiments, the edible composition further comprises at least one edible ingredient. In some cases, the edible ingredient is a solvent, such as water, carbonated water, alcohol, fruit juice, and any other commercially available beverage.

In embodiments, the edible composition comprising the protein product and having a reduced amount of recombinant cell by-product has one or more different properties relative to an equivalent edible composition without a reduced amount of recombinant cell by-product.

In embodiments, properties include density, viscosity, gel hardness, chewiness, foam capacity, foam stability, solubility, clarity, texture, foaming, whipping, exudation, gelation, clarification, coagulation, coating, crystallization control, drying, edible (edible) packaging film, finishing (finishing), flavor, fortification, freeze resistance, gloss, moisture retention, insulation, moisture retention, mouthfeel, pH stability, protein enrichment, richness, shelf life extension, texture, tenderization, texture, thickening, water binding, oil binding, browning, emulsification, nitrogen to carbon ratio, and/or antimicrobial activity. In some cases, the different property includes a desired increase in the property, or the different property includes a desired decrease in the property.

One aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and a recombinant cell by-product, wherein the recombinant cell by-product is an Extracellular Polysaccharide (EPS) or an off-flavor component; processing the composition under conditions that separate the recombinant protein from EPS or off-flavor components; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of EPS and/or collecting the isolated EPS, thereby obtaining an EPS product having a reduced amount of recombinant protein; and formulating an edible composition comprising the protein product or EPS product. In the method, the processing steps include: i) A resin that is reversibly attached to the recombinant protein and is substantially unattached to the EPS, ii) an enzyme that digests the recombinant protein or EPS, iii) an adsorbent that is reversibly attached to the EPS and is substantially unattached to the recombinant protein, and/or iv) a flocculant that is attached to the EPS and is substantially unattached to the recombinant protein.

Method for collecting recombinant cell byproducts

In any of the aspects or embodiments disclosed herein, the method may further comprise the step of collecting the isolated recombinant cell by-product. In some cases, the method further comprises the step of concentrating and/or purifying the isolated recombinant cell by-product, thereby obtaining an EPS product having a reduced amount of recombinant protein.

An exemplary method for producing a composition comprising a recombinant protein and a recombinant cell by-product and separating the recombinant protein from the recombinant cell by-product is shown in fig. 5. The methods of the present aspect can be used to collect any extracellular product produced during the fermentation process.

In one aspect, the present disclosure provides an Extracellular Polysaccharide (EPS) product produced by any of the methods disclosed herein.

In another aspect, the present disclosure provides an edible composition comprising any of the Extracellular Polysaccharide (EPS) products disclosed herein.

In embodiments, the edible composition further comprises at least one edible ingredient.

In some embodiments, the edible composition is used as a food product.

In various embodiments, the EPS provides nutritional supplements to the consumer.

In embodiments, EPS improves consumer gastrointestinal health by preventing pathogen binding to consumer gut cells.

In some embodiments, EPS improves consumer gastrointestinal health by promoting beneficial intestinal microbiomes.

One aspect of the present disclosure is a method for preparing an edible composition. The method comprises the following steps: obtaining a composition comprising a recombinant protein and a recombinant cell by-product, wherein the recombinant cell by-product is an Extracellular Polysaccharide (EPS); processing the composition under conditions that separate the recombinant protein from EPS; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of EPS and/or collecting the isolated EPS, thereby obtaining an EPS product having a reduced amount of recombinant protein; and formulating an edible composition comprising the protein product or EPS product. In the method, the processing steps include: i) A resin that is reversibly attached to the recombinant protein and is substantially unattached to the EPS, ii) an enzyme that digests the recombinant protein or EPS, iii) an adsorbent that is reversibly attached to the EPS and is substantially unattached to the recombinant protein, and/or iv) a flocculant that is attached to the EPS and is substantially unattached to the recombinant protein.

Features of the methods of the present disclosure

In embodiments, the ratio of recombinant cell by-product to recombinant protein in the composition comprising recombinant protein and recombinant cell by-product is from about 1:3 to about 3:1. In some cases, the ratio is about 1:1.

In some embodiments, the protein product having a reduced amount of recombinant cell by-product comprises at least a 50% reduction in the amount of recombinant cell by-product relative to a composition comprising the recombinant protein and recombinant cell by-product. In some cases, the protein product has at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of recombinant cell by-products relative to a composition comprising the recombinant protein and recombinant cell by-products.

In various embodiments, less than about 10% by weight of the protein product constitutes recombinant cell by-products. In some cases, less than about 5% by weight of the protein product constitutes recombinant cell by-products.

In embodiments, when size exclusion chromatography is used, EPS or off-flavor components are generally not separable from recombinant proteins.

In some embodiments, the EPS or off-flavor component is naturally a component of the cell wall of the recombinant cell. In some cases, EPS or off-flavor components present in a composition comprising recombinant protein and recombinant cell by-products are secreted from the recombinant cells, rather than being incorporated into the cell walls of the recombinant cells.

In embodiments, EPS comprises mannose. In some cases, the EPS further comprises N-acetylglucosamine and/or glucose.

In some embodiments, EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose, as analyzed by gas chromatography coupled with mass spectrometry. EPS can be quantified using a method employing pb binding columns. Analytical HyperREZ XP Pb ++ columns (8 um,300× 7.7mm,Thermofisher Sci.) can be used for measurement, which are eluted with water on a UltiMate 3000 system (Thermofisher sci.) operating at a flow rate of 0.6mL/min and monitored with a refractive index detector.

In embodiments, EPS is mannan.

In some embodiments, the recombinant cell is a cell that expresses and/or secretes EPS and is selected from a fungal cell, such as a filamentous fungus or yeast, a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

In the context of a number of embodiments of the present invention, the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces cerevisiae, a species of the genus Schizosaccharomyces pombe, a species of the genus Pichia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride. In some cases, the fungus is a pichia species. In some cases, the pichia species is saccharomyces falciparum or saccharomyces pastoris.

In embodiments, the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive. In some cases, the enzyme is pepsinogen or pepsin.

In some embodiments, the protein is an egg white protein. Eggs are almost essential food components in the world. Eggs and egg components are a vast market. Eggs provide high protein and nutritional components and are considered complete foods in combination with milk. However, eggs have a limited shelf life and are prone to infectious pathogens. People around the world, particularly children, are diagnosed with food allergies or eating restrictions prohibit them from eating eggs. In addition, in order to improve productivity of industrial-scale production of eggs, the use of growth hormone has been introduced in addition to the conditions of raising chickens which are not humane. Current egg substitutes have major limitations. None of the products expands the application to foaming and gelling. The product composition in the package is unstable over time.

In some cases, the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, an avidin, an ovalbumin-related protein X, or an ovalbumin-related protein Y, and any combination thereof. In many cases, the egg white protein is OVA, OVD, OVT or OVL. In some cases, the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to naturally occurring egg white protein in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

Any of the compositions or methods disclosed herein are applicable to any of the compositions or methods disclosed herein. In other words, any aspect or embodiment described herein may be combined with any other aspect or embodiment as disclosed herein.

Definition of the definition

The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, e.g., limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to practice in the art. Alternatively, "about" may mean a range of up to 20%, up to 15%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, in particular in relation to biological systems or methods, the term may mean within a certain order of magnitude of a value, preferably within 5 times of a value, and more preferably within 2 times of a value. Where a particular value is described in the present disclosure and claims, unless otherwise indicated, the term "about" shall be assumed to mean within an acceptable error range for that particular value.

Sequence identity, such as for the purpose of evaluating percent complementarity, may be measured by any suitable alignment algorithm, including, but not limited to, the Needleman-Wunsch algorithm (see, e.g., the EMBOSS Needle aligners available on ebi.ac/Tools/psa/EMBOSS _needle/nucleic. Html, optionally with default settings), the BLAST algorithm (see, e.g., the BLAST alignment Tools available on blast.ncbi.nlm.nih.gov/blast.cgi, optionally with default settings), and the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligners available on ebi.ac/Tools/psa/EMBOSS _water/nucleic. Htrnl, optionally with default settings). Any suitable parameter of the selection algorithm may be used to evaluate the optimal alignment, including default parameters.

The term "birds" includes both domesticated birds and non-domesticated birds such as wild animals and the like. Birds include, but are not limited to, poultry, birds (fowl), waterfowl, shotgun, ratites (e.g., non-flying birds), chickens (Gallus gallus domesticus)), quails, turkeys, ducks, ostriches (african ostrich (Struthio camelus)), somatid ostrich (Somali ostrich) (somatid ostrich (Struthio molybdophanes)), geese, gull, guinea fowl, pheasants, emu (Dromaius novaehollandiae), american ostrich (AMERICAN RHEA) (big-American ostrich (RHEA AMERICANA)), darwin's rhea (small-American ostrich (RHEA PENNATA)) and kiwi (kiwi). Tissues, cells, and their progeny of biological entities obtained in vivo or cultured in vitro are also contemplated. Birds can lay eggs.

As used herein, the term "edible composition" or "edible product" refers to a composition or product that comprises a recombinant protein or a composition comprising a recombinant protein and other ingredients and that can be consumed (e.g., by eating, chewing, drinking, tasting, ingesting, or swallowing). Edible products include food products, beverage products, dietary supplements, food additives, pharmaceuticals, and hygiene products, as non-limiting examples. Food products include, but are not limited to, baked goods (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelets, custard, pasta, noodles, kola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, cream, foam, meringue, frosting, desserts, marshmallow, marzipan, soup, condiments, sauce, spices, dairy products, and condiments. Beverage products include, but are not limited to, soft drinks, flavored waters, juices, sports drinks, energy drinks, smoothies, milkshakes, alcoholic beverages (e.g., wine, sake, beer, spirits), cocktails, liqueurs, carbonated beverages, caffeine-containing beverages, coffee, cocoa, tea, egg and milk drinks, and dairy beverages. Dietary supplements include multivitamins, whole food supplements, dietary supplements, herbal supplements, protein mixtures, weighting agents, ready-to-drink proteins, protein bars, protein milkshakes, protein powders, protein pellets, protein isolates, energy bars, energy gels, energy chews, energy formulas, endurance formulas, energy supplements, nutritional supplements, sports nutritional supplements, infant formulas (e.g., powders or liquids), and dietary substitutes. Consumers of the edible products or edible compositions are any animal, including domestic animals (e.g., livestock) and humans.

Processing the edible product to form a processed edible product may include, but is not limited to, freezing, cooling, heating, baking, boiling, scalding, packaging, canning, bleaching, enriching, drying, pressing, grinding, mixing, precooking (parcooking), cooking (cooking), proofing (proofing), curing, cutting, slicing, dicing, crushing, shredding, chopping, shaking, stoning, spiraling, rolling, juicing, staining, filtering, kneading, stirring, beating, whipping, grinding, filling (stuffing), peeling, seed removal, smoking, curing (curing), curing (salping), preserving, curing, fermenting, homogenizing, pasteurizing, sterilizing, stabilizing, blending (blending), gelatinizing (pureeing), strengthening, refining, hydrogenation, aging, extending shelf life, or adding enzymes.

As used herein, the term "solvent" refers to a liquid that can be mixed with or used to solubilize a composition or one or more components of a composition, such as a protein. Non-limiting examples of solvents include water, ethanol, and isopropanol. The solvent may be potable. The solvent may be water. Non-limiting examples of water include purified water, distilled water, double distilled water, deionized water, distilled deionized water, potable water, well water, tap water, spring water, bottled water, carbonated water, mineral water, flavored water, or any combination thereof. The solvent may be a combination of two or more different solvents.

Additional embodiments:

Embodiment 1. A process for preparing an edible composition, the process comprising the steps of: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an anionic resin that is reversibly attached to the recombinant protein and that is substantially unattached to the more than one recombinant cell by-product; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Embodiment 2. The method according to embodiment 1, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product is a cell culture medium.

Embodiment 3. The method of embodiment 1 or embodiment 2, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium comprising recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 4. The method of embodiment 1, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 5. The method of any of embodiments 1 to 4, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

Embodiment 6. The method of embodiment 5, wherein the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

Embodiment 7. The method of any of embodiments 1 to 6, wherein the anion resin is a strong anion exchange resin or a weak anion exchange resin.

Embodiment 8. The method of any of embodiments 1 to 7, wherein the anionic resin is one or more of the following: capto Q resin, DEAE type weak anion exchanger, resin with trimethylaminoethyl group, resin with triethylaminoethyl group, resin with quaternary amine group.

Embodiment 9. The method of any of embodiments 1 to 8, wherein the anionic resin is a component of a chromatography system.

Embodiment 10. The method of embodiment 9, wherein the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column.

Embodiment 11. The method of embodiment 9, wherein the chromatography system is operated in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography method (e.g., equilibration, loading, elution, and washing) occur simultaneously.

Embodiment 12. The method of embodiment 11, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

Embodiment 13. The method of any of embodiments 4 to 12, wherein the composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

Embodiment 14. The method of embodiment 13 wherein the treatment to remove small non-protein molecules comprises the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

Embodiment 15. The method of embodiment 13 or embodiment 14, wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

Embodiment 16. The method according to any one of embodiments 1 to 15, further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

Embodiment 17. The method of embodiment 16, wherein the protein-containing composition having preferred pH and/or ionic conditions is further heat treated and/or dried.

Embodiment 18. The method of any of embodiments 1 to 15, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further heat treated and/or dried.

Embodiment 19. The method of embodiment 17 or embodiment 18, wherein heat treatment separates the recombinant protein and the off-flavor component, wherein heat is applied at a temperature and for a duration such that the off-flavor component volatilizes and the gaseous off-flavor component is removable.

Embodiment 20. The method of embodiment 19, wherein the vacuum is applied simultaneously with the application of heat and facilitates removal of gaseous off-flavor components.

Embodiment 21. The method of any of embodiments 1 to 20 wherein the odor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

Embodiment 22. The method of any of embodiments 1 to 21, wherein the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

Embodiment 23. The method of any of embodiments 17 to 22, wherein the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃, during the heat treatment.

Embodiment 24. The method of any of embodiments 17 to 23, wherein the method comprises stirring during the heat treatment.

Embodiment 25. The method of any one of embodiments 17 to 24, wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

Embodiment 26. The method of any of embodiments 1 to 25, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step, e.g., comprising adding hydrogen peroxide.

Embodiment 27. The method of any of embodiments 1 to 26, wherein the ratio of recombinant cell by-product to recombinant protein in the composition comprising recombinant protein and more than one recombinant cell by-product is about 1:3 to about 3:1.

Embodiment 28. The method of embodiment 27, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to the composition comprising the recombinant protein and more than one recombinant cell by-product.

Embodiment 29. The method of embodiment 28, wherein less than about 10% of the weight of the protein product comprises recombinant cell byproducts.

Embodiment 30. The method of any of embodiments 1 to 29, wherein less than about 5% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 31. The method of any of embodiments 1 to 30, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises an off-flavor component.

Embodiment 32. The method of any of embodiments 1 to 31, wherein off-flavor components in the protein product are virtually undetectable to a standard consumer.

Embodiment 33. The method of any one of embodiments 1 to 32, wherein EPS is generally inseparable from recombinant protein when size exclusion chromatography is used.

Embodiment 34. The method of any one of embodiments 1 to 33, wherein EPS is naturally a component of the cell wall of recombinant cells.

Embodiment 35. The method of any of embodiments 1 to 34, wherein the EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

Embodiment 36. The method of any one of embodiments 1 to 35, wherein the EPS comprises mannose.

Embodiment 37 the method of any one of embodiments 1 to 36, wherein the EPS further comprises N-acetylglucosamine and/or glucose.

Embodiment 38. The method of any one of embodiments 1 to 37, wherein EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose as analyzed by gas chromatography coupled with mass spectrometry.

Embodiment 39. The method of any of embodiments 1 to 38, wherein the EPS comprises an α (1, 6) -linked backbone having α (1, 2) -linked branches and/or α (1, 3) -linked branches.

Embodiment 40. The method of any one of embodiments 1 to 39, wherein EPS is mannan.

Embodiment 41. The method of any of embodiments 1 to 40, wherein the recombinant cells expressing the recombinant protein and more than one recombinant cell by-product are selected from the group consisting of fungal cells, such as filamentous fungi or yeast, bacterial cells, plant cells, insect cells, or mammalian cells.

Embodiment 42. The method according to any one of embodiments 1 to 41, wherein the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces, a species of the genus Schizosaccharomyces pombe, a species of the genus Phaffia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

Embodiment 43. The method of embodiment 41 or embodiment 42, wherein the fungus is a pichia species.

Embodiment 44. The method of embodiment 43, wherein the Pichia species is Fabry colt or Pasteur colt.

Embodiment 45. The method of any of embodiments 1 to 44, wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

Embodiment 46. The method of embodiment 45 wherein the enzyme is pepsinogen or pepsin.

Embodiment 47. The method of embodiment 45, wherein the protein is an egg white protein.

Embodiment 48. The method of embodiment 47, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

Embodiment 49. The method of embodiment 47 or embodiment 48, wherein the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an egg white protein naturally produced in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

Embodiment 50. The method of any of embodiments 1 to 49, wherein the edible composition comprising the protein product comprises a food product, a beverage product, or a dietary supplement.

Embodiment 51. The method of embodiment 50 wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, candy, marshmallow, soup, condiments, sauce, spice, dairy products, and flavoring.

Embodiment 52. The method of embodiment 50 wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg and dairy beverage.

Embodiment 53. The method of embodiment 50 wherein the dietary supplement comprises a multivitamin, a whole food supplement, a dietary supplement, an herbal supplement, a protein mixture, a weighting agent, a ready-to-drink protein, a protein bar, a protein milkshake, a protein powder, a protein pellet, a protein isolate, an energy bar, an energy gel, an energy chew, an energy formula, a endurance formula, an energy supplement, a nutritional supplement, a sports nutritional supplement, an infant formula (e.g., a powder or a liquid), and a meal replacement.

Embodiment 54 the method of any one of embodiments 1 to 53, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) One or more cation exchange resins reversibly attached to the recombinant protein and not substantially attached to the EPS, ii) an enzyme that digests the recombinant protein or EPS, iii) an adsorbent reversibly attached to the EPS and not substantially attached to the recombinant protein, and/or iv) a flocculant attached to the EPS and not substantially attached to the recombinant protein.

Embodiment 55. An edible composition obtained by the method of any one of embodiments 1 to 54.

Embodiment 56. A method for preparing an edible composition, the method comprising the steps of: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises one or more cation exchange resins that are reversibly attached to the recombinant protein and that are substantially unattached to the more than one recombinant cell by-product; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Embodiment 57 the method of embodiment 56, wherein the one or more cation exchange resins comprise a strong cation exchange resin, e.g., a sulfopropyl-type resin, a sulfomethyl-type resin, or a sulfonate-type resin, and/or a weak cation exchange resin, e.g., a carboxymethyl-type resin.

Embodiment 58 the method of embodiment 56 or embodiment 57, wherein the one or more cation exchange resins comprise polystyrene divinylbenzene, polymethacrylate, or cellulose or crosslinked dextran or crosslinked agarose, or an inorganic material coated with a hydrophilic polymer.

Embodiment 59. The method of any one of embodiments 56 to 58, wherein the one or more cation exchange resins have a particle size of about 50 μm and about 200 μm and/or have a protein binding capacity of about 50g protein/L to about 100g protein/L resin.

Embodiment 60. The method of any one of embodiments 56 to 59, wherein the one or more cation exchange resins comprises Cytiva Capto S, HP20, relizorb SP400, SEPRAGEN S, SP20, and/or Mitsubishi Relizorb EXE349.

Embodiment 61. The method of any of embodiments 56-60, wherein the processing step comprises two cationic resins, wherein the two cationic resins are in a ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.

Embodiment 62. The method of embodiment 61, wherein the two resins are SP400 and SEPRAGEN S and are in a ratio of about 3:1, e.g., 2.75:1.25.

Embodiment 63 the method of any of embodiments 56 to 62, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 64 the method of any one of embodiments 56-63, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product has a pH less than the isoelectric point (pI) of the recombinant protein by reducing the pH of the composition comprising the recombinant protein and the more than one recombinant cell by-product.

Embodiment 65. The method of any of embodiments 56-64, wherein the one or more cationic resins are components of a chromatography system, wherein the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column.

Embodiment 66. The method of any of embodiments 56-64, wherein the one or more cationic resins are components of a chromatography system, wherein the chromatography system is operated in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps (e.g., equilibration, loading, elution, and washing) in the chromatography method occur simultaneously.

Embodiment 67. The method of embodiment 66, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

Embodiment 68 the method of any of embodiments 62-67, wherein the composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

Embodiment 69. The method of embodiment 68, wherein the treatment to remove small non-protein molecules comprises the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

Embodiment 70. The method of embodiment 68 or embodiment 69, wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

Embodiment 71 the method of any one of embodiments 56 to 70, further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

Embodiment 72. The method of embodiment 71, wherein the protein-containing composition having preferred pH and/or ionic conditions is further heat treated and/or dried.

Embodiment 73 the method of any one of embodiments 56 to 70, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further heat treated and/or dried.

Embodiment 74. The method of embodiment 72 or embodiment 73, wherein heat treatment separates the recombinant protein from the off-flavor component, wherein heat is applied at a temperature and for a duration such that the off-flavor component volatilizes and the gaseous off-flavor component is removable.

Embodiment 75. The method of embodiment 74, wherein the vacuum is applied simultaneously with the application of heat and the vacuum facilitates removal of gaseous off-flavor components.

Embodiment 76 the method of any of embodiments 56-75 wherein the odor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

Embodiment 77 the method of any one of embodiments 56 to 76, wherein the odor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

Embodiment 78 the method of any one of embodiments 72 to 77, wherein the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃, during the heat treatment.

Embodiment 79 the method of any of embodiments 72-78, wherein the method comprises stirring during the heat treatment.

Embodiment 80. The method of any one of embodiments 72 to 79, wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

Embodiment 81 the method of any one of embodiments 56 to 80, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step, e.g., comprising adding hydrogen peroxide.

Embodiment 82 the method of any one of embodiments 56 to 81, wherein the ratio of recombinant cell by-product to recombinant protein in the composition comprising recombinant protein and more than one recombinant cell by-product is about 1:3 to about 3:1.

Embodiment 83. The method of embodiment 82, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to the composition comprising the recombinant protein and more than one recombinant cell by-product.

Embodiment 84. The method of embodiment 83, wherein less than about 10% of the weight of the protein product comprises recombinant cell byproducts.

Embodiment 85 the method of any one of embodiments 56 to 84, wherein less than about 5% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 86 the method of any one of embodiments 56 to 85, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises an off-flavor component.

Embodiment 87 the method of any of embodiments 56-86, wherein off-flavor components in the protein product are virtually undetectable to a standard consumer.

Embodiment 88 the method of any one of embodiments 56-87, wherein EPS is generally inseparable from recombinant protein when size exclusion chromatography is used.

Embodiment 89 the method of any of embodiments 56-88, wherein EPS is naturally a component of the cell wall of a recombinant cell.

Embodiment 90 the method of any one of embodiments 56-89, wherein the EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

Embodiment 91 the method of any of embodiments 56-90, wherein the EPS comprises mannose.

Embodiment 92 the method of any of embodiments 56-91, wherein the EPS further comprises N-acetylglucosamine and/or glucose.

Embodiment 93 the method of any one of embodiments 56-92, wherein EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose as determined by gas chromatography coupled with mass spectrometry.

Embodiment 94 the method of any one of embodiments 56 to 93 wherein the EPS comprises an alpha (1, 6) -linked backbone having alpha (1, 2) -linked branches and/or alpha (1, 3) -linked branches.

Embodiment 95 the method of any one of embodiments 56 to 94, wherein EPS is mannan.

Embodiment 96 the method of any one of embodiments 56 to 95, wherein the recombinant cells expressing the recombinant protein and more than one recombinant cell by-product are selected from fungal cells, such as filamentous fungi or yeast, bacterial cells, plant cells, insect cells, or mammalian cells.

Embodiment 97 the method of any of embodiments 56-96, wherein the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces, a species of the genus Schizosaccharomyces pombe, a species of the genus Phaffia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

Embodiment 98. The method of embodiment 96 or embodiment 97, wherein the fungus is a pichia species.

Embodiment 99. The method of embodiment 98, wherein the Pichia species is Fabry colt or Barceira.

Embodiment 100 the method of any one of embodiments 56 to 99, wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

Embodiment 101. The method of embodiment 100, wherein the enzyme is pepsinogen or pepsin.

Embodiment 102. The method of embodiment 100, wherein the protein is an egg white protein.

Embodiment 103. The method of embodiment 102, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

Embodiment 104. The method of embodiment 102 or embodiment 103, wherein the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an naturally occurring egg white protein in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

Embodiment 105 the method of any one of embodiments 56 to 104, wherein the edible composition comprising a protein product comprises a food product, a beverage product, or a dietary supplement.

Embodiment 106. The method of embodiment 105, wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, candy, marshmallow, soup, condiments, sauce, spice, dairy products, and flavoring.

Embodiment 107. The method of embodiment 105 wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg and milk liquor, and dairy beverage.

Embodiment 108. The method of embodiment 105 wherein the dietary supplement comprises a multivitamin, a whole food supplement, a dietary supplement, an herbal supplement, a protein mixture, a weighting agent, a ready-to-drink protein, a protein bar, a protein milkshake, a protein powder, a protein pellet, a protein isolate, an energy bar, an energy gel, an energy chew, an energy formula, a endurance formula, an energy supplement, a nutritional supplement, a sports nutritional supplement, an infant formula (e.g., a powder or a liquid), and a meal replacement.

Embodiment 109 the method of any one of embodiments 56 to 108, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) An anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an enzyme that digests the recombinant protein or EPS, iii) an adsorbent reversibly attached to the EPS and substantially unattached to the recombinant protein, and/or iv) a flocculant attached to the EPS and substantially unattached to the recombinant protein.

Embodiment 110. An edible composition obtained by the method of any one of embodiments 56 to 109.

Embodiment 111 a method for preparing an edible composition comprising the steps of: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises a flocculant that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Embodiment 112. The method of embodiment 111, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium.

Embodiment 113 the method of embodiment 111 or embodiment 112, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium comprising recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 114 the method of any one of embodiments 111 to 113, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

Embodiment 115. The method of embodiment 114, wherein the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

Embodiment 116 the method of any one of embodiments 111 to 115, wherein the flocculant is added to a medium comprising recombinant cells that secrete recombinant proteins and more than one recombinant cell by-product.

Embodiment 117 the method of any of embodiments 111-116, wherein once the flocculant is attached to one or more components of more than one recombinant cell byproduct, the flocculant is separated from the recombinant protein.

Embodiment 118 the method of embodiment 117, wherein the flocculant is separated from the recombinant protein with a filter, a filtration device, and/or by centrifugation when the flocculant is attached to one or more components of more than one recombinant cell byproduct.

Embodiment 119 the method of embodiment 118, further comprising replenishing the culture medium with a flocculant again.

Embodiment 120 the method of any one of embodiments 111 to 119, wherein a flocculant is provided to the biomass separation feed tank and one or more components of more than one recombinant cell byproduct while the spent biomass comprising the recombinant cells is removed.

Embodiment 121 the method of any one of embodiments 111 to 119, wherein the flocculant is provided after removing spent biomass comprising recombinant cells.

Embodiment 122. The method of embodiment 121, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 123 the method of any one of embodiments 111 to 122 wherein the flocculant is an anionic flocculant or a neutral flocculant.

Embodiment 124 the method of any of embodiments 111-123, wherein the flocculant is Tramfloc, tramfloc 109, tramfloc 110, tramfloc 111 or Tramfloc 120, magnafloc 333, magnafloc 355 or Gusmer divan, dupont Polyox, celite 545, bentonite BE125, DIAION HPA25L, 85% deacetylated chitosan, EZ DE, ultra-pure diatomaceous earth or Relizorb SP400.

Embodiment 125 the method of any of embodiments 111-124 wherein a flocculant is disposed in the column.

Embodiment 126 the method of any one of embodiments 111 to 125, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises a chromatography system.

Embodiment 127. The method of embodiment 126, wherein the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column, or the chromatography system is operated in continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography method (e.g., equilibration, loading, elution, and washing) occur simultaneously.

Embodiment 128 the method of embodiment 127, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

Embodiment 129 the method of any of embodiments 121-128, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

Embodiment 130. The method of embodiment 129, wherein the treatment to remove the small non-protein molecules comprises a step of concentrating a composition comprising the recombinant protein and more than one recombinant cell by-product.

Embodiment 131. The method of embodiment 129 or embodiment 130, wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

Embodiment 132 the method of any one of embodiments 111 to 131, further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

Embodiment 133. The method of embodiment 132, wherein the protein-containing composition having the preferred pH and/or ionic conditions is further heat treated and/or dried.

Embodiment 134 the method of any one of embodiments 111 to 131, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further heat treated and/or dried.

Embodiment 135. The method of embodiment 133 or embodiment 134, wherein the heat treatment separates the recombinant protein from the odor component, wherein heat is applied at a temperature and for a duration such that the odor component volatilizes and the gaseous odor component is removable.

Embodiment 136. The method of embodiment 135, wherein the vacuum is applied simultaneously with the application of heat, and the vacuum facilitates removal of gaseous off-flavor components.

Embodiment 137 the method of any of embodiments 111-136 wherein the odor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

Embodiment 138 the method of any one of embodiments 111 to 137, wherein the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

The method according to any one of embodiments 133-138, wherein the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃ during the heat treatment.

Embodiment 140 the method of any of embodiments 133-139, wherein the method comprises stirring during the heat treatment.

Embodiment 141 the method of any one of embodiments 133-140, wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

Embodiment 142 the method of any one of embodiments 111 to 141, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step.

Embodiment 143 the method of embodiment 142, wherein the oxidizing step comprises adding hydrogen peroxide.

Embodiment 144 the method of any one of embodiments 111 to 143, wherein the ratio of recombinant cell by-product to recombinant protein in the composition comprising recombinant protein and more than one recombinant cell by-product is about 1:3 to about 3:1.

Embodiment 145. The method of embodiment 144, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to the composition comprising the recombinant protein and more than one recombinant cell by-product.

Embodiment 146. The method of embodiment 145, wherein less than about 10% of the weight of the protein product comprises recombinant cell byproducts.

Embodiment 147 the method of any one of embodiments 111 to 146, wherein less than about 5% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 148 the method of any of embodiments 111 through 147, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises an off-flavor component.

Embodiment 149 the method of any one of embodiments 111 to 148, wherein off-flavor components in the protein product are virtually undetectable to a standard consumer.

Embodiment 150 the method of any one of embodiments 111 to 149, wherein EPS is generally inseparable from recombinant protein when size exclusion chromatography is used.

Embodiment 151 the method of any one of embodiments 111 to 150, wherein EPS is naturally a component of the cell wall of a recombinant cell.

Embodiment 152 the method of any one of embodiments 111-151, wherein EPS has an apparent size of about 13kDa to about 27kDa as characterized according to a size exclusion chromatography column.

Embodiment 153 the method of any one of embodiments 111-152, wherein EPS comprises mannose.

Embodiment 154 the method of any one of embodiments 111-153, wherein EPS further comprises N-acetylglucosamine and/or glucose.

Embodiment 155 the method of any one of embodiments 111-154, wherein EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose as determined by gas chromatography coupled with mass spectrometry.

Embodiment 156 the method of any one of embodiments 111 to 155, wherein the EPS comprises an alpha (1, 6) -linked backbone having alpha (1, 2) -linked branches and/or alpha (1, 3) -linked branches.

Embodiment 157 the method of any of embodiments 111-156, wherein EPS is mannan.

Embodiment 158 the method of any one of embodiments 111 to 157, wherein the recombinant cell expressing the recombinant protein and more than one recombinant cell by-product is selected from a fungal cell, such as a filamentous fungus or a yeast, a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

Embodiment 159 the method of any one of embodiments 111 to 158, wherein the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces, a species of the genus Schizosaccharomyces pombe, a species of the genus Phaffia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

Embodiment 160. The method of embodiment 158 or embodiment 159, wherein the fungus is a pichia species.

Embodiment 161. The method of embodiment 160, wherein the pichia species is f.coltsfoot or s.pastoris.

Embodiment 162 the method of any of embodiments 111 to 161, wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

Embodiment 163. The method of embodiment 162 wherein the enzyme is pepsinogen or pepsin.

Embodiment 164. The method of embodiment 163 wherein the protein is an egg white protein.

Embodiment 165. The method of embodiment 164, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

Embodiment 166. The method of embodiment 164 or embodiment 165, wherein the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an egg white protein naturally produced in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

Embodiment 167 the method of any of embodiments 111 to 166, wherein the edible composition comprising the protein product comprises a food product, a beverage product, or a dietary supplement.

Embodiment 168 the method of embodiment 167 wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, dessert, marshmallow, soup, condiments, sauce, spice, dairy products, and flavoring.

Embodiment 169. The method of embodiment 167, wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg and dairy beverage.

Embodiment 170. The method of embodiment 167, wherein the dietary supplement comprises a multivitamin, a whole food supplement, a dietary supplement, an herbal supplement, a protein mixture, a weighting agent, a ready-to-drink protein, a protein bar, a protein milkshake, a protein powder, a protein pellet, a protein isolate, an energy bar, an energy gel, an energy chew, an energy formula, a endurance formula, an energy supplement, a nutritional supplement, a sports nutritional supplement, an infant formula (e.g., a powder or a liquid), and a meal replacement.

Embodiment 171 the method of any one of embodiments 111 to 170, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) A cationic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, iii) an enzyme that digests the recombinant protein or EPS, and/or iv) an adsorbent attached to the EPS and substantially unattached to the recombinant protein.

Embodiment 172. An edible composition obtained by the method of any one of embodiments 111 to 171.

Embodiment 173 a method for preparing an edible composition comprising the steps of: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an adsorbent that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Embodiment 174 the method of embodiment 173, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product is a cell culture medium.

Embodiment 175. The method of embodiment 173 or embodiment 174, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium comprising recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 176 the method of any one of embodiments 173 to 175, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

Embodiment 177. The method of embodiment 176, wherein the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

Embodiment 178 the method of any one of embodiments 173-177, wherein the adsorbent is added to a medium comprising recombinant cells that secrete the recombinant protein and more than one recombinant cell by-product.

The method of any one of embodiments 173-178, wherein the adsorbent is separated from the recombinant protein once the adsorbent is attached to one or more components of more than one recombinant cell by-product.

Embodiment 180. The method of embodiment 179, wherein the adsorbent is separated from the recombinant protein with a filter, filtration apparatus, and/or by centrifugation when the adsorbent is attached to one or more components of more than one recombinant cell byproduct.

Embodiment 181. The method of embodiment 180, further comprising replenishing the medium with an adsorbent again.

Embodiment 182 the method of any one of embodiments 173-181 wherein the adsorbent is provided to the biomass separation feed tank and one or more components of more than one recombinant cell byproduct while the spent biomass comprising the recombinant cells is removed.

Embodiment 183 the method of any of embodiments 173-181, wherein the adsorbent is provided after the removal of the spent biomass comprising the recombinant cells.

Embodiment 184. The method of embodiment 183 wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 185 the method of any one of embodiments 173 to 184, wherein the adsorbent comprises a resin and/or a hydrophobic adsorbent comprising, for example, a methacrylate or silica backbone, or is a DEAE-type weak anion exchanger.

Embodiment 186 the method of any of embodiments 173-185 wherein the adsorbent is Dow Amberlite SD2, mitsubishi Diaion HP, celite 545, bentonite BE125, DIAION HPA25L, 85% deacetylated chitosan, EZ DE, ultra pure diatomaceous earth, or Relizorb SP400.

Embodiment 187 the method of any of embodiments 173-186 wherein the adsorbent is disposed in, for example, a column and operated in batch mode with an axial flow column or radial flow column or centrifugal column, or by using a membrane chromatography column.

Embodiment 188 the method of any one of embodiments 173-187, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises a chromatography system.

Embodiment 189. The method of embodiment 188, wherein the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column, or the chromatography system is operated in continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography method (e.g., equilibration, loading, elution, and washing) occur simultaneously.

Embodiment 190 the method of embodiment 189, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

Embodiment 191 the method of any of embodiments 183 to 190, wherein a composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

Embodiment 192. The method of embodiment 191 wherein the treatment to remove small non-protein molecules comprises the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

Embodiment 193 the method of embodiment 191 or embodiment 192 wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

Embodiment 194 the method of any of embodiments 173-193 further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

Embodiment 195. The method of embodiment 194, wherein the protein-containing composition having preferred pH and/or ionic conditions is further heat treated and/or dried.

Embodiment 196 the method of any of embodiments 173-193, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further heat treated and/or dried.

Embodiment 197 the method of embodiment 195 or embodiment 196, wherein heat treatment separates the recombinant protein and the off-flavor component, wherein heat is applied at a temperature and for a duration such that the off-flavor component volatilizes and the gaseous off-flavor component is removable.

Embodiment 198. The method of embodiment 197 wherein a vacuum is applied simultaneously with the application of heat and the vacuum facilitates removal of gaseous off-flavor components.

Embodiment 199 the method of any of embodiments 173-198, wherein the odor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

Embodiment 200 the method of any of embodiments 173 to 199, wherein the odor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

Embodiment 201 the method of any one of embodiments 195 to 200, wherein the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃, during the heat treatment.

Embodiment 202 the method of any one of embodiments 195-201, wherein the method comprises stirring during the heat treatment.

Embodiment 203 the method of any one of embodiments 195 to 202 wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

Embodiment 204 the method of any of embodiments 173-203, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step.

Embodiment 205. The method of embodiment 204 wherein the oxidizing step comprises adding hydrogen peroxide.

Embodiment 206 the method of any one of embodiments 173-205, wherein the ratio of recombinant cell by-product to recombinant protein in the composition comprising recombinant protein and more than one recombinant cell by-product is about 1:3 to about 3:1.

Embodiment 207. The method of embodiment 206, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to the composition comprising the recombinant protein and more than one recombinant cell by-product.

Embodiment 208. The method of embodiment 207 wherein less than about 10% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 209 the method of any of embodiments 173-208, wherein less than about 5% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 210 the method of any one of embodiments 173 to 209, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises an off-flavor component.

Embodiment 211 the method of any one of embodiments 173 to 210, wherein off-flavor components in the protein product are virtually undetectable to a standard consumer.

Embodiment 212 the method of any one of embodiments 173-211, wherein EPS is generally inseparable from recombinant protein when size exclusion chromatography is used.

Embodiment 213 the method of any one of embodiments 173 to 212, wherein EPS is naturally a component of the cell wall of a recombinant cell.

The method of any one of embodiments 173 to 213, wherein the EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

Embodiment 215 the method of any of embodiments 173-214 wherein EPS comprises mannose.

Embodiment 216 the method of any one of embodiments 173 to 215, wherein the EPS further comprises N-acetylglucosamine and/or glucose.

Embodiment 217 the method of any one of embodiments 173 to 216, wherein EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose as according to gas chromatography analysis in combination with mass spectrometry.

Embodiment 218 the method of any one of embodiments 173 to 217 wherein the EPS comprises an alpha (1, 6) -linked backbone having alpha (1, 2) -linked branches and/or alpha (1, 3) -linked branches.

Embodiment 219 the method of any one of embodiments 173 to 218, wherein EPS is mannan.

Embodiment 220 the method of any one of embodiments 173-219, wherein the recombinant cell expressing the recombinant protein and more than one recombinant cell by-product is selected from a fungal cell, such as a filamentous fungus or yeast, a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

Embodiment 221 the method of any one of embodiments 173 to 220, wherein the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces, a species of the genus Schizosaccharomyces pombe, a species of the genus Phaffia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

Embodiment 222. The method of embodiment 220 or embodiment 221 wherein the fungus is a pichia species.

Embodiment 223. The method of embodiment 222, wherein the Pichia species is Fabry colt or Barceira.

Embodiment 224 the method of any one of embodiments 173 to 223 wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

Embodiment 225 the method of embodiment 224 wherein the enzyme is pepsinogen or pepsin.

Embodiment 226. The method of embodiment 225, wherein the protein is an egg white protein.

Embodiment 227 the method of embodiment 226, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

Embodiment 228. The method of embodiment 226 or embodiment 227, wherein the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an egg white protein naturally produced in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

Embodiment 229 the method of any of embodiments 173-228 wherein the edible composition comprising a protein product comprises a food product, a beverage product, or a dietary supplement.

Embodiment 230. The method of embodiment 229 wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, candy, marshmallow, soup, condiments, sauce, spice, dairy products, and flavoring.

Embodiment 231 the method of embodiment 229 wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg and milk wine, and dairy beverage.

Embodiment 232. The method of embodiment 229 wherein the dietary supplement comprises a multivitamin, a whole food supplement, a dietary supplement, an herbal supplement, a protein mixture, a weighting agent, a ready-to-drink protein, a protein bar, a protein milkshake, a protein powder, a protein pellet, a protein isolate, an energy bar, an energy gel, an energy chew, an energy formula, a endurance formula, an energy supplement, a nutritional supplement, a sports nutritional supplement, an infant formula (e.g., a powder or a liquid), and a meal replacement.

Embodiment 233 the method of any one of embodiments 173-232, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) A cationic resin that is reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an anionic resin that is reversibly attached to the recombinant protein and substantially unattached to the EPS, iii) an enzyme that digests the recombinant protein or EPS, and/or iv) a flocculant that is attached to the EPS and substantially unattached to the recombinant protein.

Embodiment 234 an edible composition obtained by the method of any one of embodiments 173 to 233.

Embodiment 235 a process for preparing an edible composition comprising the steps of: obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component; processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product, wherein the processing step comprises digesting the recombinant protein or an enzyme that digests EPS; collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of more than one recombinant cell by-product; and formulating an edible composition comprising the protein product.

Embodiment 236 the method of embodiment 235, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium.

Embodiment 237 the method of embodiment 235 or embodiment 236, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium comprising recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 238 the method of embodiment 235 wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

Embodiment 239 the method of any of embodiments 235-238, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

Embodiment 240. The method of embodiment 239 wherein the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

Embodiment 241 the method of embodiment 239, wherein the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is adjusted to achieve a pH greater than the pI of the recombinant protein.

Embodiment 242. The method of embodiment 240, wherein the pH of the composition comprising the recombinant protein and the more than one recombinant cell by-product is about 6.

Embodiment 243 the method of any of embodiments 235-238, wherein the composition comprising the recombinant protein and more than one recombinant cell by-product has a pH that is less than the isoelectric point (pI) of the recombinant protein.

Embodiment 244. The method of embodiment 243, wherein the pH of the composition comprising the recombinant protein and more than one recombinant cell by-product is achieved by lowering the pH.

Embodiment 245 the method of any one of embodiments 235 to 244, wherein the enzyme that digests the recombinant protein is pepsin or trypsin.

Embodiment 246. The method of embodiment 245, wherein the digested recombinant protein permeates through an ultrafiltration system having a10 kDa membrane.

Embodiment 247 the method of any one of embodiments 235 to 244, wherein the enzyme that digests EPS is mannanase, cellulase, or glucanase.

Embodiment 248. The method of embodiment 247, wherein the undigested recombinant protein is concentrated by an ultrafiltration system having a 5kDa membrane.

Embodiment 249 the method of any of embodiments 235-248, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises a chromatography system.

Embodiment 250. The method of embodiment 249, wherein the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column.

Embodiment 251. The method of embodiment 249 wherein the chromatography system is operated in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography method (e.g., equilibration, loading, elution, and washing) occur simultaneously.

Embodiment 252. The method of embodiment 251, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

Embodiment 253 the method of any one of embodiments 245 to 252, wherein a composition comprising recombinant protein and more than one recombinant cell by-product is pre-treated to remove spent biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

Embodiment 254 the method of embodiment 253 wherein the treatment to remove small non-protein molecules comprises the step of concentrating a composition comprising recombinant protein and more than one recombinant cell by-product.

Embodiment 255 the method of embodiment 253 or embodiment 254 wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

Embodiment 256 the method of any one of embodiments 235-255, further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

Embodiment 257. The method of embodiment 256, wherein the protein-containing composition having preferred pH and/or ionic conditions is further heat treated and/or dried.

Embodiment 258 the method of any one of embodiments 235 to 255, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product and/or the protein product having a reduced amount of the more than one recombinant cell by-product is further heat treated and/or dried.

Embodiment 259. The method of embodiment 257 or embodiment 258, wherein heat treatment separates the recombinant protein and the off-flavor component, wherein heat is applied at a temperature and for a duration such that the off-flavor component volatilizes and the gaseous off-flavor component is removable.

Embodiment 260 the method of embodiment 259, wherein a vacuum is applied simultaneously with the application of heat, and the vacuum facilitates removal of gaseous off-flavor components.

Embodiment 261 the method of any one of embodiments 235-260, wherein the odor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

Embodiment 262 the method of any one of embodiments 235 to 261 wherein the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

The method of any one of embodiments 257 to 262, wherein the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising recombinant protein and more than one recombinant cell by-product, and/or the protein product having a reduced amount of more than one recombinant cell by-product is up to 80 ℃, e.g., about 50 ℃ to about 60 ℃ during the heat treatment.

Embodiment 264 the method of any of embodiments 257-263, wherein the method comprises stirring during the heat treatment.

Embodiment 265 the method of any one of embodiments 257 to 264, wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of more than one recombinant cell by-product.

Embodiment 266 the method of any of embodiments 235-265, wherein the composition comprising recombinant protein and more than one recombinant cell by-product and/or the protein product having a reduced amount of more than one recombinant cell by-product is further subjected to an oxidation step.

Embodiment 267. The method of embodiment 266 wherein the oxidizing step comprises adding hydrogen peroxide.

Embodiment 268 the method of any one of embodiments 235 to 267 wherein the ratio of recombinant cell by-product to recombinant protein in the composition comprising recombinant protein and more than one recombinant cell by-product is from about 1:3 to about 3:1.

Embodiment 269 the method of embodiment 268, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to a composition comprising the recombinant protein and more than one recombinant cell by-product.

Embodiment 270 the method of embodiment 269, wherein less than about 10% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 271 the method of any one of embodiments 235-270, wherein less than about 5% by weight of the protein product comprises recombinant cell byproducts.

Embodiment 272. The method of any of embodiments 235-271, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises an off-flavor component.

Embodiment 273 the method of any of embodiments 235-272, wherein off-flavor components in the protein product are virtually undetectable to a standard consumer.

Embodiment 274 the method of any of embodiments 235-273, wherein EPS is generally inseparable from recombinant protein when size exclusion chromatography is used.

Embodiment 275 the method of any one of embodiments 235 to 274, wherein EPS is naturally a component of the cell wall of a recombinant cell.

The method of any one of embodiments 235-275, wherein EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

Embodiment 277 the method of any of embodiments 235-276, wherein EPS comprises mannose.

Embodiment 278 the method of any one of embodiments 235-277, wherein EPS further comprises N-acetylglucosamine and/or glucose.

Embodiment 279 the method of any one of embodiments 235-278, wherein EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose, as determined by gas chromatography coupled with mass spectrometry.

Embodiment 280 the method of any one of embodiments 235-279, wherein the EPS comprises an alpha (1, 6) -linked backbone having alpha (1, 2) -linked branches and/or alpha (1, 3) -linked branches.

Embodiment 281 the method of any of embodiments 235-280, wherein EPS is mannan.

Embodiment 282 the method of any of embodiments 235 to 281, wherein the recombinant cell expressing the recombinant protein and more than one recombinant cell by-product is selected from a fungal cell, such as a filamentous fungus or yeast, a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

Embodiment 283 the method of any of embodiments 235 to 282, wherein the recombinant cell type is selected from the group consisting of an species of the genus Saccharomyces, a species of the genus Kluyveromyces, a species of the genus Pichia, a species of the genus Saccharomyces, a species of the genus Schizosaccharomyces pombe, a species of the genus Phaffia yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria species, endocarpium parasiticum, escherichia coli, bacillus subtilis, bacillus anthracis, endocarpium species, and Bacillus subtilis Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

Embodiment 284. The method of embodiment 282 or embodiment 283, wherein the fungus is a pichia species.

Embodiment 285. The method of embodiment 284, wherein the Pichia species is F.falciparum or F.pastoris.

Embodiment 286 the method of any of embodiments 235-285, wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

Embodiment 287. The method of embodiment 286, wherein the enzyme is pepsinogen or pepsin.

Embodiment 288 the method of embodiment 287, wherein the protein is an egg white protein.

Embodiment 289 the method of embodiment 288, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoid, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

Embodiment 290 the method of embodiment 288 or embodiment 289, wherein the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to an egg white protein naturally produced in an avian such as chicken, quail, turkey bald, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

Embodiment 291 the method of any of embodiments 235-290, wherein the edible composition comprising a protein product comprises a food product, a beverage product, or a dietary supplement.

Embodiment 292. The method of embodiment 291 wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetables, tofu, cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, toffee, emulsion, foam, meringue, frosting, candy, marshmallow, soup, condiments, sauce, spice, dairy products, and flavoring.

Embodiment 293. The method of embodiment 291 wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg milk, and dairy beverage.

Embodiment 294 the method of embodiment 291 wherein the dietary supplement comprises a multivitamin, a whole food supplement, a dietary supplement, an herbal supplement, a protein mixture, a weighting agent, a ready-to-drink protein, a protein bar, a protein milkshake, a protein powder, a protein pellet, a protein isolate, an energy bar, an energy gel, an energy chew, an energy formula, a endurance formula, an energy supplement, a nutritional supplement, a sports nutritional supplement, an infant formula (e.g., powder or liquid), and a meal replacement.

Embodiment 295 the method of any one of embodiments 235 to 294, wherein processing the composition under conditions that separate the recombinant protein from more than one recombinant cell by-product further comprises one or more of: i) A cationic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, iii) a flocculant attached to the EPS and substantially unattached to the recombinant protein, and/or iv) an adsorbent attached to the EPS and substantially unattached to the recombinant protein.

Embodiment 296 an edible composition obtained by the method of any one of embodiments 235 to 295.

Additionally, the above embodiments described herein may be combined with any other embodiment as disclosed above.

Examples

Example 1: use of resins for the preparation of protein products with reduced amounts of recombinant cell byproducts

The methods of the present disclosure (e.g., as shown in fig. 1) are used to prepare protein products with reduced amounts of recombinant cell byproducts. In this example, recombinant egg white proteins were used.

Table 1 (below) shows typical compositions comprising recombinant protein and recombinant cell byproducts before and after using a resin (e.g., chromatography) based purification method to reduce the amount of recombinant cell byproducts.

Table 1: comparison of physicochemical properties of the concentrated protein before and after the purification step.

	Concentrated proteins	Purified proteins
			Protein (% w/w)	53	98
EPS(％w/w)	47	2

Purified proteins are also used to exhibit unique gelling properties similar to commercial egg white, thereby enabling formulation replacement of eggs in egg-based formulations.

Table 2 below shows a comparison of various additional functional properties of the concentrated composition (comprising recombinant protein and recombinant cell by-product) and purified protein product (having a reduced amount of recombinant cell by-product).

Table 2: comparison of functional properties of the concentrated proteins before and after the purification step.

	Concentrated proteins	Purified proteins
			Gel hardness	High height	Low and low
Masticatory properties	High height	Low and low
			Foam capacity	High height	High height
Foam stability	Low and low	High height

Notably, removal of recombinant cell byproducts (e.g., EPS) reduces the gel firmness and chewiness of the exemplary edible composition/food product counterparts.

Table 3 below shows a comparison of various additional functional properties of the concentrated compositions (comprising recombinant proteins and recombinant cell byproducts) and commercially available egg substitutes.

Table 3: comparison of functional Properties of concentrated compositions (comprising recombinant proteins and recombinant cell byproducts) with commercial egg white proteins

	Concentrated proteins	Egg white protein
			Gel hardness	High height	High height
Masticatory properties	High height	High height
			Foam capacity	High height	High height

Surprisingly, it has been found that the gelling properties of the concentrated composition (comprising recombinant protein and recombinant cell by-products) are comparable to egg white powder, indicating a significant contribution of impurities to the gelling. Further, the foaming and foam retention properties are shown in table 2. Thus, for high foam applications, it may be preferable to specifically regulate the amount of recombinant cell byproducts in the edible composition.

The purified recombinant ovalbumin product is combined with varying amounts of recombinant cell byproducts (e.g., EPS or off-flavor components) to determine changes in the product properties.

Resins having sulfopropyl, sulfomethyl, sulfonic acid groups can be used in this process. The backbone is typically a non-protein binding material such as methacrylate or cellulose, typically having a particle size between 50um and 200 um. Ligand density will accommodate protein binding capacity between 50g protein/L resin and 100g protein/L resin.

Example 2: another use of a resin for the preparation of a protein product with reduced amounts of recombinant cell byproducts

Another method of the present disclosure (e.g., as shown in fig. 1 or fig. 7) is used to prepare a protein product with reduced amounts of recombinant cell byproducts. In this example, recombinant egg white proteins were used.

Pichia pastoris strains derived from the historian Phillips Petroleum strain NRRL Y-11430 were designed to produce a non-methanol use (mutM) phenotype that was transformed to express the exemplary protein (here, ovomucoid) and a strong methanol inducible promoter. These transformed strains were further modified by the addition of surface displaying enzymes that reduced complex carbohydrates to filterable sizes. Sequencing confirmed that the strain did not contain any antibiotic markers or replication sequences from prokaryotic vector sources.

The resulting strain was grown under fermentation conditions under high density growth conditions at about pH 5. After about 36 hours of growth under fermentation conditions, the pH was raised to about pH6 and expression of the exemplary proteins was induced by adding methanol to the culture. The fermentation broth (fermentation broth) was centrifuged (using a bench centrifuge-Avant J18 rotor Bechman Coulter) to remove cells. This was followed by filtration of the supernatant using a 0.2 μm hollow fiber membrane to remove host proteins and cell debris. The protein solution was then concentrated to over 30g/L protein using a 5kDa ultrafiltration membrane, and diafiltered extensively to remove most of the organic and inorganic impurities. The resulting protein concentrate was adjusted to pH 3.5 using citrate and loaded onto a chromatographic column. The column was packed with cation exchange resin (SP 400, mitsubishi Chemicals, japan). The chromatography step was performed in a downflow mode at about 22℃using AKTA Explorer 900 (GE HEALTHCARE LIFE SCIENCES) and Unicorn interface software (version 5.11). Chromatographic methods generally consist of an equilibration step, a loading (flow-through) step, a washing step to remove unbound protein, an elution step to remove product, a clean-in-place (CIP) step, and a regeneration step. The elution column volumes at each step and the buffers used are shown in table 4 below.

Table 4: a list of buffer and column volumes required in a typical process.

Elution profile is shown in figure 12.

In this and other embodiments, the following terminology is used: the "equilibrium" is the preloaded column, the "flow through" is the material that passes through the column as the UF diafiltered concentrate passes through the column, and is shown on the gel as lane 3 with minimal product loss, "elution" is the protein-rich fraction of interest (e.g., in the gel of fig. 14, lane 4) and is the largest peak in the chromatogram, and "CIP" is the wash solution elution.

In an alternative method, the column is packed with a unique mixture of two resins. SP400 (Mitsubishi Chemicals, japan) and SEPRAGEN S (Sepragen, california) are in a ratio of 2.75:1.25. Buffer composition and column volume were maintained as shown in table 4. The elution profile is shown in figure 13.

Example 3: anionic resins for preparing protein products with reduced amounts of recombinant cell byproducts

In example 2, the column was packed with cationic resin. In this example, the column was packed with anion exchange Capto Q resin (CYTIVA CHEMICALS). Buffer composition and column volume were maintained as shown in table 5. Note that the pH of the feed was not adjusted here for significant simplification of the process. Note that the pH of the feed was not adjusted here for significant simplification of the process.

Table 5: a list of buffer and column volumes required in a typical process.

Elution profile is shown in fig. 14A, and gel showing protein fraction is shown in fig. 14B.

Resins having a trimethylaminoethyl group, a triethylaminoethyl group, a quaternary amine group can be used in the method described in this example.

Example 4: use of adsorbents or flocculants for the preparation of protein products with reduced amounts of recombinant cell byproducts

The protein concentrate prepared according to example 3 above was further tested with a variety of adsorbents and flocculants. The procedure used was as follows: 0.05g of the test adsorbent or flocculant was weighed out in a 50mL falcon tube. To the test adsorbent or flocculant was added 5mL of protein concentrate. The tube was then placed on a roller shaker (roller shaker) for 1 hour, followed by centrifugation at 3214g for 30min. The supernatants were then tested for absorbance, protein, and EPS. Each material was tested in duplicate. The adsorbents and flocculants tested are listed below: control (pH 6), control (pH 4), celite 545, bentonite BE125, DIAION HPA25L, 85% deacetylated chitosan, EZ DE (diatomaceous earth), ultra-pure diatomaceous earth, relizorb SP400 (pH 4). The absorbance spectra of this study are shown in fig. 15. Notably Relizorb is not functional because it binds to proteins and not to impurities at pH 4.0. At higher pH, this resin is expected to elute everything.

Additional data are shown in fig. 16 and 17. These figures show that HPA25L and chitosan adsorb more EPS than the control (black) and hardly adsorb protein, whereas sp 400 adsorbs protein at pH 4 without adsorbing EPS. Diatomaceous Earth (DE) adsorbs both protein and EPS without difference.

Example 5: application of flocculant in EPS extraction

Pichia pastoris strains grow under fermentation conditions under high density growth conditions at about pH 5. After about 36 hours of growth under fermentation conditions, the pH is raised to about pH 6 and expression of the protein of interest is induced by adding methanol to the culture. At the end of the fermentation, flocculant was added to the broth at a concentration of 10mg/L to 100 mg/L. The culture broth was mixed and maintained at room temperature or 4 ℃ for 6 hours to allow complete utilization of the glucose in the medium and the flocculant to function. A coagulant may be added at this point. The fermentation broth was centrifuged (using a bench top centrifuge-Avant J18 rotor Bechman Coulter) to remove cells and EPS-like compounds. This was followed by filtration of the supernatant using a 0.2 μm hollow fiber membrane to remove host proteins and cell debris. The protein solution was then concentrated to over 30g/L protein using a 5kDa ultrafiltration membrane, and diafiltered extensively to remove most of the organic and inorganic impurities. The resulting mixture is subjected to microfiltration or heat treatment for the final microorganism reduction step prior to drying. The steps of the method are shown in fig. 4 or fig. 10.

Example 6: use of enzymatic digestion of EPS

Pichia pastoris strains grow under fermentation conditions under high density growth conditions at about pH 5. After about 36 hours of growth under fermentation conditions, the pH is raised to about pH 6 and expression of the protein of interest is induced by adding methanol to the culture. At this point, EPS degrading enzymes such as glucanase are added to the reactor. This will degrade the EPS molecules as they are secreted in the system. The final fermentation broth was centrifuged (using a bench top centrifuge-Avant J18 rotor Bechman Coulter) to remove cells. This was followed by filtration of the supernatant using a 0.2 μm hollow fiber membrane to remove host proteins and cell debris. The protein solution was then concentrated to over 30g/L protein using a 5kDa ultrafiltration membrane, and diafiltered extensively to remove most of the organic and inorganic impurities. EPS degrading enzymes are removed with the biomass in a centrifuge and/or MF step. The resulting mixture is subjected to microfiltration or heat treatment for the final microorganism reduction step prior to drying. The steps of the method are shown in fig. 2 or fig. 8.

Example 7: use of adsorbent for extracting EPS

Pichia pastoris strains grow under fermentation conditions under high density growth conditions at about pH 5. After about 36 hours of growth under fermentation conditions, the pH is raised to about pH 6 and expression of the protein of interest is induced by adding methanol to the culture. At the end of the fermentation, the culture broth is mixed and maintained at room temperature or 4 ℃ for 6 hours to allow complete utilization of the glucose in the medium. The fermentation broth was centrifuged (using a bench top centrifuge-Avant J18 rotor Bechman Coulter) to remove cells. The centrate (centrate) from the centrifuge is then passed through a filter pre-coated with an adsorbent to remove EPS-like compounds. If a filter is used, the adsorbent is suspended in the water and passed through the filter, forming a uniform layer on the filter. This is followed by passage of the centrate through a filter. This can also be done with a column packed with the same adsorbent. After this step, the supernatant was filtered using a 0.2 μm hollow fiber membrane to remove host proteins and cell debris. The protein solution was then concentrated to over 30g/L protein using a 5kDa ultrafiltration membrane, and diafiltered extensively to remove most of the organic and inorganic impurities. The resulting mixture is subjected to microfiltration or heat treatment for the final microorganism reduction step prior to drying. The steps of the method are shown in fig. 3 or fig. 9.

Example 8: removal of adverse characteristics

The protein concentrate obtained in the manner described in example 2 was then precipitated by adding ammonium sulphate to the concentrate solution to reach a concentration of about 40% w/v. The pellet was then centrifuged (using a bench top centrifuge-Avant J18 rotor Bechman Coulter) to remove protein. The protein was then resuspended in 10% w/v solution using DI water. The solution was then diafiltered with DI water to a final conductivity of less than 1 mS/cm. The diafiltered protein was then micro-filtered through a 0.2 μm membrane and freeze-dried. These proteins are known as "small-scale preparations".

For large scale fermentation, at the end of the fermentation, the broth is cooled to 8 ℃ to slow down the metabolism of the yeast. Prior to centrifugation, the culture broth was diluted to reach a packed cell volume of 25% v/v. The yeast cells were then removed by centrifugation and the supernatant was stored and transferred to the next step. This step is completed within 8 hours of completion of the fermentation and may begin before cooling is complete. For this purpose, a suitable disk centrifuge with a sufficiently large surface area and solids capacity is used. The overall OD600 measurement of the supernatant indicating good separation is preferably <0.9AU. The centrifugate was collected by 0.2 μm filtration for further clarification. The 0.2 μm filtration will operate in tangential flow mode with both the concentration and diafiltration steps. During the concentration step, the retentate (retentate) volume was reduced by about 6-9×. To achieve higher yields, the retentate was continuously diafiltered with 10 diafiltration volumes (diavolume) of water. Permeate from 0.2 μm TFF was concentrated from its initial volume to a protein concentration of 6-8 x to about 50 g/L. The final retentate was dark green in color. The retentate was then diafiltered with 6-8DV DI water. The diafiltered retentate was then sterile filtered using a 0.2 μm MF filter and spray dried at an inlet temperature of about 165 ℃ and an outlet temperature of no more than 80 ℃. The membrane may be a hydrophilic polyvinyl sulfone designed for protein applications. Throughout the process, the temperature was maintained at 10 ℃. These proteins are known as "large-scale preparations".

The above procedure resulted in a dark green solution pre-drying. In order to decolorize and deodorize it, an oxidation step may be used. The pH of the retentate prior to diafiltration was reduced to 4 using 85% v/v phosphoric acid. Then, 35% v/v hydrogen peroxide was slowly added to saturate the final solution to a 3% v/v hydrogen peroxide mixture. The mixture was then kept in the tank for 6 hours while slowly mixing to prevent the foam from flowing out. The pH was then changed back to 6 using concentrated sodium hydroxide, followed by diafiltration, sterile filtration and drying. These protein samples are referred to as "example a" samples.

Samples from example a were analyzed by GC-MS for end of fermentation and end of process. The main flavors and flavor compounds observed are listed in table 6 below:

table 6: list of compounds that caused significant flavor/odor at the end of fermentation and purification.

The pH of the resulting protein retentate from both small gauge molded articles and large scale articles was adjusted and loaded onto a chromatographic column. The column was packed with cation exchange resin (SP 400, mitsubishi Chemicals, japan). The chromatography step was performed in a downflow mode at about 22℃using AKTA Explorer 900 (GE HEALTHCARE LIFE SCIENCES) and Unicorn interface software (version 5.11). Chromatographic methods generally consist of an equilibration step, a loading (flow-through) step, a washing step to remove unbound protein, an elution step to remove product, a clean-in-place (CIP) step, and a regeneration step. The elution column volumes at each step and the buffers used are shown in table 4 above and the elution profile is shown in figure 12.

The eluted fraction from the column is then concentrated and diafiltered to remove the elution buffer salt. The stream was then microfiltered using a 0.2 μm membrane and dried. These samples are referred to as "example B" samples.

The oxidized and non-oxidized samples were then tested by GC-MS using a polar Stabilwax-DA column. The sample preparation used was SPME and the analysis consisted of MS data and a human olfactory odour test. Referring to table 7, the following are the results for small-scale protein preparations:

Table 7: GC MS data of samples (small scale preparations) generated in laboratory settings using oxidation and chromatographic methods.

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Referring to table 8, the following are the results for the large-scale protein preparation:

table 8 GC MS data of samples (small scale preparations) generated in laboratory apparatus using oxidation and chromatographic methods.

When comparing the example a sample with the example B sample, most of the compounds listed in table 7 were reduced. This is even more evident in table 8 in the case of large scale articles.

The example B samples produced above were further reprocessed using various treatments to examine the improvement in sensory properties. The test was processed as follows:

Ethanol washing: the spray-dried protein powder was resuspended in 10% v/v ethanol solution to achieve a solids concentration of 50 g/L. The solution was stirred using a magnetic stirrer at ambient temperature for 1 hour and then diafiltered with 4-5DV DI water over a 5kDa membrane. The retentate was sterile filtered and dried.

Ion Exchange (IEX): the spray dried protein powder was resuspended in DI water to achieve a solids concentration of 50 g/L. The procedure described in example 5 was repeated with the protein solution.

Heating and vacuum: the spray dried protein powder was resuspended in DI water to achieve a solids concentration of 50 g/L. The protein solution was then heated to 50-58 ℃ and maintained under a low vacuum (75-150 torr) for 1 hour. The solution was then sterile filtered and dried.

IEX, heat and vacuum: this is an orthogonal method combining multiple purification methods together. The product from example 5 was then resuspended in DI water to achieve a solids concentration of 50 g/L. The protein solution was then heated to 50-58 ℃ and maintained under a low vacuum (75-150 torr) for 1 hour. The solution was then sterile filtered and dried.

Sensory analysis of these samples based on the appearance, mouthfeel and aftertaste of the 6% w/v solution in water is analyzed in fig. 18. The ion exchange process of the non-oxidized article produced the best form of reprocessing control. The center of the figure represents the sensory characteristics (profile) of water.

Incorporated by reference

All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. In the event that publications and patents or patent applications incorporated by reference contradict the disclosure contained in this specification, this specification is intended to supersede and/or take precedence over any such contradictory material.

Claims

1. A method for preparing an edible composition, the method comprising the steps of:

Obtaining a composition comprising a recombinant protein and more than one recombinant cell by-product, wherein the recombinant cell by-product comprises an Extracellular Polysaccharide (EPS) and an off-flavor component;

Processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an anionic resin that is reversibly attached to the recombinant protein and that is substantially unattached to the more than one recombinant cell by-product;

Collecting the isolated recombinant protein, thereby obtaining a protein product having a reduced amount of the more than one recombinant cell by-product; and

An edible composition comprising the protein product is formulated.

2. The method of claim 1, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium.

3. The method of claim 1 or claim 2, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is a cell culture medium comprising recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

4. The method of claim 1, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

5. The method of any one of claims 1-4, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product has a pH greater than the isoelectric point (pI) of the recombinant protein.

6. The method of claim 5, wherein the pH of the composition comprising the recombinant protein and the more than one recombinant cell by-product is not adjusted to achieve a pH greater than the pI of the recombinant protein.

7. The method of any one of claims 1 to 6, wherein the anion resin is a strong anion exchange resin or a weak anion exchange resin.

8. The method of any one of claims 1 to 7, wherein the anionic resin is one or more of the following: capto Q resin, DEAE type weak anion exchanger, resin with trimethylaminoethyl group, resin with triethylaminoethyl group, resin with quaternary amine group.

9. The method of any one of claims 1 to 8, wherein the anionic resin is a component of a chromatographic system.

10. The method of claim 9, wherein the chromatography system is operated in batch mode with an axial flow column or a radial flow column or a centrifugal column, or by using a membrane chromatography column.

11. The method of claim 9, wherein the chromatography system is operated in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps in the chromatography process (e.g., equilibration, loading, elution, and washing) occur simultaneously.

12. The method of claim 11, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

13. The method of any one of claims 4 to 12, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

14. The method of claim 13, wherein the treatment to remove small non-protein molecules comprises the step of concentrating the composition comprising the recombinant protein and the more than one recombinant cell by-product.

15. The method of claim 13 or claim 14, wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

16. The method of any one of claims 1 to 15, further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

17. The method of claim 16, wherein the protein-containing composition having preferred pH and/or ionic conditions is further heat treated and/or dried.

18. The method of any one of claims 1 to 15, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product and/or the protein product having a reduced amount of the more than one recombinant cell by-product is further heat treated and/or dried.

19. The method of claim 17 or claim 18, wherein the heat treatment separates the recombinant protein and the off-flavor component, wherein heat is applied at a temperature and for a duration such that the off-flavor component is volatilized and gaseous off-flavor component is removable.

20. The method of claim 19, wherein a vacuum is applied simultaneously with the application of heat, and the vacuum facilitates removal of the gaseous malodorous components.

21. The method of any one of claims 1 to 20, wherein the off-flavor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

22. The method of any one of claims 1 to 21, wherein the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

23. The method according to any one of claims 17 to 22, wherein the temperature of the protein-containing composition with preferred pH and/or ionic conditions, the composition comprising the recombinant protein and the more than one recombinant cell by-product and/or the protein product with reduced amounts of the more than one recombinant cell by-product is up to 80 ℃, such as about 50 ℃ to about 60 ℃ during the heat treatment.

24. The method of any one of claims 17 to 23, wherein the method comprises agitation during the heat treatment.

25. The method of any one of claims 17 to 24, wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of the more than one recombinant cell by-product.

26. The method of any one of claims 1 to 25, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product and/or the protein product having a reduced amount of the more than one recombinant cell by-product is further subjected to an oxidation step, e.g., comprising the addition of hydrogen peroxide.

27. The method of any one of claims 1-26, wherein the ratio of the recombinant cell by-product to recombinant protein in the composition comprising the recombinant protein and the more than one recombinant cell by-product is about 1:3 to about 3:1.

28. The method of claim 27, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to the composition comprising the recombinant protein and the more than one recombinant cell byproduct.

29. The method of claim 28, wherein less than about 10% by weight of the protein product comprises recombinant cell byproducts.

30. The method of any one of claims 1-29, wherein less than about 5% by weight of the protein product comprises recombinant cell byproducts.

31. The method of any one of claims 1 to 30, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises the off-flavor component.

32. The method of any one of claims 1 to 31, wherein the off-flavor component in the protein product is virtually undetectable to a standard consumer.

33. The method of any one of claims 1 to 32, wherein the EPS is generally inseparable from the recombinant protein when size exclusion chromatography is used.

34. The method of any one of claims 1 to 33, wherein the EPS is naturally a component of the cell wall of a recombinant cell.

35. The method of any one of claims 1 to 34, wherein the EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

36. The method of any one of claims 1-35, wherein the EPS comprises mannose.

37. The method of any one of claims 1-36, wherein the EPS further comprises N-acetylglucosamine and/or glucose.

38. The method of any one of claims 1-37, wherein the EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose according to gas chromatography analysis in combination with mass spectrometry.

39. The method of any one of claims 1 to 38, wherein the EPS comprises an a (1, 6) -linked backbone having a (1, 2) -linked branches and/or a (1, 3) -linked branches.

40. The method of any one of claims 1 to 39, wherein the EPS is mannan.

41. The method of any one of claims 1to 40, wherein the recombinant cells expressing the recombinant protein and the more than one recombinant cell by-product are selected from fungal cells, such as filamentous fungi or yeast, bacterial cells, plant cells, insect cells, or mammalian cells.

42. The method according to any one of claims 1 to 41, wherein the recombinant cell type is selected from the group consisting of a species of the genus Arxula (Arxula spp.), a species of the genus adenylate (Arxula adeninivorans), a species of the genus Kluyveromyces (Kluyveromyces spp.), a species of the genus Kluyveromyces lactis (Kluyveromyces lactis), a species of the genus favus (Komagataella phaffii), a species of the genus Pichia (Pichia spp.), a Pichia angusta (Pichia angusta), a Pichia pastoris (Pichia pastoris), a species of the genus Saccharomyces (Saccharomyces spp.), a species of the genus Saccharomyces (Saccharomyces cerevisiae), a species of the genus Schizosaccharomyces (Schizosaccharomyces spp.), a species of the genus Schizosaccharomyces pombe (Schizosaccharomyces pombe), a species of the genus Yarrowia (Yarrowia spp.); yarrowia lipolytica (Yarrowia lipolytica), agaricus species (Agaricus spp.), agaricus bisporus (Agaricus bisporus), aspergillus species (Aspergillus spp.), aspergillus awamori (Aspergillus awamori), aspergillus fumigatus (Aspergillus fumigatus), aspergillus nidulans (Aspergillus nidulans), aspergillus niger (Aspergillus niger), aspergillus oryzae (Aspergillus oryzae), bacillus subtilis (Bacillus subtilis), anthrax species (Colletotrichum spp.), colletotrichum anthrax (Colletotrichum gloeosporiodes), colletotrichum species (Endothia spp.), inner housing (Endothia parasitica), escherichia coli (ESCHERICHIA COLI), fusarium spp), fusarium graminearum (Fusarium graminearum), fusarium solani (Fusarium solani), mucor species (Mucor spp.), fusarium spp, mucor miehei (Mucor miehei), mucor miehei (Mucor pusillus), myceliophthora species (Myceliophora spp.), myceliophthora thermophila (Myceliophthora thermophila), neurospora species (Neurospora spp.), neurospora crassa (Neurospora crassa), penicillium species (Penicillium spp.), penicillium shamendocina (Penicillium camemberti), penicillium cinerea (Penicillium canescens), penicillium chrysogenum (Penicillium chrysogenum), penicillium amortium (Talaromyces) (Penicillium (Talaromyces) emersonii), penicillium funiculosum (Penicillium funiculosum), penicillium purpurogenum (Penicillium purpurogenum), penicillium romyces (Penicillium roqueforti), pleurotus species (Pleurotus spp.), pleurotus (Pleurotus ostreatus), pseudomonas species (Pseudomonas spp.), rhizomucor spp.), rhizopus (Rhizomucor miehei), rhizopus (Rhizomucor pusillus), rhizopus (Rhizopus, rhizopus (3774), trichoderma viridis (3742), trichoderma viride (3742.

43. The method of claim 41 or claim 42, wherein the fungus is a Pichia species.

44. The method of claim 43, wherein the Pichia species is Fabry-Perot or Pasteurella (Komagataella pastoris).

45. The method of any one of claims 1 to 44, wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

46. The method of claim 45, wherein the enzyme is pepsinogen or pepsin.

47. The method of claim 45, wherein the protein is an egg white protein.

48. The method of claim 47, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoin, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

49. The method of claim 47 or claim 48, wherein the egg white protein has a sequence at least 80% identical (e.g., about 85%, 90%, or 95% identical) to naturally occurring egg white protein in an avian such as chicken, quail, turkey baldness, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

50. The method of any one of claims 1 to 49, wherein the edible composition comprising the protein product comprises a food product, a beverage product, or a dietary supplement.

51. The method of claim 50, wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, battercake, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetable, tofu (tofu), tofu (bean curd), cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, custard, emulsion, foam, meringue, frosting, dessert, marshmallow, soup, condiments, sauce, spices, dairy products, and condiments.

52. The method of claim 50, wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg milk, and dairy beverage.

53. The method of claim 50, wherein the dietary supplement comprises a multivitamin, a whole food supplement, a dietary supplement, an herbal supplement, a protein mixture, a weighting agent, a ready-to-drink protein, a protein bar, a protein milkshake, a protein powder, a protein pellet, a protein isolate, an energy bar, an energy gel, an energy chew, an energy formula, a endurance formula, an energy supplement, a nutritional supplement, a sports nutritional supplement, an infant formula (e.g., powder or liquid), and a meal replacement.

54. The method of any one of claims 1-53, wherein processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product further comprises one or more of: i) One or more cation exchange resins reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an enzyme that digests the recombinant protein or the EPS, iii) an adsorbent reversibly attached to the EPS and substantially unattached to the recombinant protein, and/or iv) a flocculant attached to the EPS and substantially unattached to the recombinant protein.

55. An edible composition obtained by the method of any one of claims 1 to 54.

56. A method for preparing an edible composition, the method comprising the steps of:

Processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises one or more cation exchange resins that are reversibly attached to the recombinant protein and that are substantially unattached to the more than one recombinant cell by-product;

An edible composition comprising the protein product is formulated.

57. The method of claim 56, wherein said one or more cation exchange resins comprise a strong cation exchange resin, e.g., a sulfopropyl, sulfomethyl, or sulfogroup type resin, and/or a weak cation exchange resin, e.g., a carboxymethyl type resin.

58. The method of claim 56 or claim 57, wherein said one or more cation exchange resins comprise polystyrene divinylbenzene, polymethacrylate or cellulose or crosslinked dextran or crosslinked agarose or inorganic materials coated with hydrophilic polymers.

59. The method of any one of claims 56 to 58, wherein the one or more cation exchange resins have a particle size of about 50 μιη and about 200 μιη and/or have a protein binding capacity of about 50g protein/L resin to about 100g protein/L resin.

60. The method of any one of claims 56 to 59, wherein said one or more cation exchange resins comprises Cytiva Capto S, HP20, relizorb SP400, SEPRAGEN S, SP20, and/or Mitsubishi Relizorb EXE349.

61. The method of any one of claims 56 to 60, wherein the processing step comprises two cationic resins, wherein the two cationic resins are in a ratio of 1:5 to 5:1, 1:4 to 4:1, 1:3 to 3:1, 1:2 to 2:1, or 1:1.

62. The method of claim 61, wherein the two resins are SP400 and SEPRAGEN S and are in a ratio of about 3:1, e.g., 2.75:1.25.

63. The method of any one of claims 56-62, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is free of recombinant cells that secrete the recombinant protein and the more than one recombinant cell by-product.

64. The method of any one of claims 56-63, wherein said composition comprising said recombinant protein and said more than one recombinant cell by-product has a pH less than the isoelectric point (pI) of said recombinant protein by lowering the pH of said composition comprising said recombinant protein and said more than one recombinant cell by-product.

65. The method of any one of claims 56 to 64, wherein the one or more cationic resins are components of a chromatography system, wherein the chromatography system is operated in batch mode with an axial flow column or radial flow column or centrifugal column, or by using a membrane chromatography column.

66. The method of any one of claims 56 to 64, wherein the one or more cationic resins are components of a chromatography system, wherein the chromatography system is operated in a continuous mode comprising a plurality of parallel columns, the feed to the columns being switchable such that multiple steps (e.g., equilibration, loading, elution, and washing) in the chromatography method occur simultaneously.

67. The method of claim 66, wherein the continuous mode comprises a Simulated Moving Bed (SMB) or an ion separator (e.g.,) The system.

68. The method of any one of claims 62 to 67, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product is pre-treated to remove waste biomass comprising recombinant cells and/or pre-treated to remove small non-protein molecules.

69. The method of claim 68, wherein the treatment to remove small non-protein molecules comprises the step of concentrating the composition comprising the recombinant protein and the more than one recombinant cell by-product.

70. The method of claim 68 or claim 69, wherein the treatment to remove small non-protein molecules comprises diafiltration buffer.

71. The method of any one of claims 56 to 70, further comprising a concentration step and/or diafiltration treatment of the isolated recombinant protein to produce a protein-containing composition having preferred pH and/or ionic conditions.

72. The method of claim 71, wherein the protein-containing composition having preferred pH and/or ionic conditions is further heat treated and/or dried.

73. The method of any one of claims 56-70, wherein said composition comprising said recombinant protein and said more than one recombinant cell by-product and/or said protein product having a reduced amount of said more than one recombinant cell by-product is further heat treated and/or dried.

74. The method of claim 72 or claim 73, wherein the heat treatment separates the recombinant protein and the off-flavor component, wherein heat is applied at a temperature and for a duration such that the off-flavor component volatilizes and gaseous off-flavor component is removable.

75. The method of claim 74, wherein a vacuum is applied simultaneously with the application of heat and facilitates removal of the gaseous malodor components.

76. The method of any one of claims 56 to 75, wherein the off-flavor component is an acid, alcohol, aldehyde, aromatic, ester, or ketone.

77. The method of any one of claims 56 to 76, wherein the off-flavor component is (E) -2-nonenal; 1-dodecene; 2-ethyl-1-hexanol; 1-hexen-3-one; 1-octen-3-one; 2, 3-butanedione; 2-butanone; 2-methyl butyraldehyde; 2-methylpropionaldehyde; 2-propanone; 2-undecone; 3-methyl butyraldehyde; acetaldehyde; phenethyl alcohol; benzyl alcohol; 3-methyl-butyraldehyde; chlorotoluene; pelargonic acid; p-cresol; or 3-hydroxy-2, 4-trimethylpentyl 2-methyl-propionate.

78. The method of any one of claims 72 to 77, wherein the temperature of the protein-containing composition having preferred pH and/or ionic conditions, the composition comprising the recombinant protein and the more than one recombinant cell by-product, and/or the protein product having a reduced amount of the more than one recombinant cell by-product is up to 80 ℃, such as about 50 ℃ to about 60 ℃ during the heat treatment.

79. The method of any one of claims 72-78, wherein the method comprises agitation during the heat treatment.

80. The method of any one of claims 72 to 79, wherein the heat treating and/or drying step produces a dried protein product having a reduced amount of the more than one recombinant cell by-product.

81. The method of any one of claims 56-80, wherein the composition comprising the recombinant protein and the more than one recombinant cell by-product and/or the protein product having a reduced amount of the more than one recombinant cell by-product is further subjected to an oxidation step, e.g., comprising adding hydrogen peroxide.

82. The method of any one of claims 56-81, wherein the ratio of the recombinant cell by-product to recombinant protein in the composition comprising the recombinant protein and the more than one recombinant cell by-product is about 1:3 to about 3:1.

83. The method of claim 82, wherein the protein product has at least a 25% reduction, at least a 30% reduction, at least a 35% reduction, at least a 40% reduction, at least a 45% reduction, at least a 50% reduction, at least a 55% reduction, at least a 60% reduction, at least a 65% reduction, at least a 70% reduction, at least a 75% reduction, at least a 80% reduction, at least a 90% reduction, or at least a 95% reduction in the amount of EPS and/or the amount of off-flavor components relative to the composition comprising recombinant protein and the more than one recombinant cell byproduct.

84. The method of claim 83, wherein less than about 10% by weight of the protein product comprises recombinant cell byproducts.

85. The method of any one of claims 56-84, wherein less than about 5% by weight of said protein product comprises recombinant cell byproducts.

86. The method of any one of claims 56 to 85, wherein less than about 5%, less than about 1%, less than about 0.1%, or less than about 0.01% by weight of the protein product comprises the off-flavor component.

87. The method of any one of claims 56-86, wherein the off-flavor component in the protein product is virtually undetectable to a standard consumer.

88. The method of any one of claims 56-87, wherein the EPS is generally inseparable from the recombinant protein when size exclusion chromatography is used.

89. The method of any one of claims 56-88, wherein said EPS is naturally a component of a cell wall of a recombinant cell.

90. The method of any one of claims 56-89, wherein the EPS has an apparent size of about 13kDa to about 27kDa as characterized by a size exclusion chromatography column.

91. The method of any one of claims 56-90, wherein the EPS comprises mannose.

92. The method of any one of claims 56-91, wherein the EPS further comprises N-acetylglucosamine and/or glucose.

93. The method of any one of claims 56-92, wherein the EPS comprises about 91mol% mannose, about 5mol% n-acetylglucosamine, and about 3mol% glucose according to gas chromatography analysis in combination with mass spectrometry.

94. The method of any one of claims 56 to 93, wherein the EPS comprises an a (1, 6) -linked backbone having a (1, 2) -linked branches and/or a (1, 3) -linked branches.

95. The method of any one of claims 56-94, wherein the EPS is mannan.

96. The method of any one of claims 56-95, wherein said recombinant cell expressing said recombinant protein and said more than one recombinant cell by-product is selected from a fungal cell, such as a filamentous fungus or yeast, a bacterial cell, a plant cell, an insect cell, or a mammalian cell.

97. The method according to any one of claims 56 to 96, wherein the recombinant cell type is selected from the group consisting of Saccharomyces species, alternaria species, kluyveromyces lactis, phaffia, pichia species, pichia angusta, pichia pastoris, saccharomyces species, saccharomyces cerevisiae, schizosaccharomyces species, schizosaccharomyces pombe, yarrowia species, yarrowia lipolytica, agaricus species, agaricus bisporus, aspergillus species, aspergillus awamori, aspergillus fumigatus, aspergillus nidulans, aspergillus niger, aspergillus oryzae, bacillus subtilis, bacillus anthracis species, alternaria parasitica, escherichia coli Fusarium species, fusarium graminearum, fusarium putrescens, mucor species, mucor miehei, mucor minutissima, myceliophthora species, myceliophthora thermophila, neurospora species, neurospora crassa, penicillium species, penicillium salvinii, penicillium griseum, penicillium chrysogenum, penicillium emerald (Talaromyces), penicillium funiculosum, penicillium purpurogenum, penicillium roqueforti, pleurotus species, pseudomonas species, rhizomucor miehei, rhizomucor minutissima, rhizopus oligosporus, rhizopus oryzae, trichoderma species, trichoderma atroviride, trichoderma reesei, and Trichoderma viride.

98. The method of claim 96 or claim 97, wherein the fungus is a pichia species.

99. The method of claim 98, wherein the pichia species is f.

100. The method of any one of claims 56-99, wherein the recombinant protein is an enzyme, a nutritional protein, a food ingredient, or a food additive.

101. The method of claim 100, wherein the enzyme is pepsinogen or pepsin.

102. The method of claim 100, wherein the protein is an egg white protein.

103. The method of claim 102, wherein the egg white protein is Ovalbumin (OVA), ovomucoid (OVD), ovotransferrin (OVT), lysozyme (OVL), ovomucoin, ovoglobulin G2, ovoglobulin G3, an egg inhibitor, an egg glycoprotein, a flavoprotein, an ovomacroglobulin, an egg-solid protein, a cysteine protease inhibitor, avidin, ovalbumin-related protein X, or ovalbumin-related protein Y, and any combination thereof.

104. The method of claim 102 or claim 103, wherein the egg white protein has a sequence that is at least 80% identical (e.g., about 85%, 90%, or 95% identical) to naturally occurring egg white protein in an avian such as chicken, quail, turkey baldness, buzzy, duck, ostrich, goose, gull, guinea fowl, pheasant, or emu.

105. The method of any one of claims 56-104, wherein said edible composition comprising said protein product comprises a food product, a beverage product, or a dietary supplement.

106. The method of claim 105, wherein the food product comprises a baked good (e.g., cake, muffin, cookie, bread, bagel, pastry, doughnut), omelet, battercake, pasta, noodles, cola, wafer, dough, batter, cookie dough, meat roll, meatball, hamburger, animal feed, fruit, vegetable, tofu (tofu), tofu (bean curd), cheese, seafood, meat, ice cream, mayonnaise, custard, pudding, custard, emulsion, foam, meringue, frosting, dessert, marshmallow, soup, condiments, sauce, spices, dairy products, and condiments.

107. The method of claim 105, wherein the beverage product comprises a soft drink, flavored water, fruit juice, sports drink, energy drink, smoothie, milkshake, alcoholic beverage (e.g., wine, sake, beer, spirits), cocktail, liqueur, carbonated beverage, caffeine-containing beverage, coffee, cocoa, tea, egg milk, and dairy beverage.

108. The method of claim 105, wherein the dietary supplement comprises multivitamins, whole food supplements, dietary supplements, herbal supplements, protein mixtures, weighting agents, ready-to-drink proteins, protein bars, protein milkshakes, protein powders, protein pellets, protein isolates, energy bars, energy gels, energy chews, energy formulas, endurance formulas, energy supplements, nutritional supplements, sports nutritional supplements, infant formulas (e.g., powders or liquids), and dietary substitutes.

109. The method of any one of claims 56-108, wherein processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product further comprises one or more of: i) An anionic resin reversibly attached to the recombinant protein and substantially unattached to the EPS, ii) an enzyme that digests the recombinant protein or the EPS, iii) an adsorbent reversibly attached to the EPS and substantially unattached to the recombinant protein, and/or iv) a flocculant attached to the EPS and substantially unattached to the recombinant protein.

110. An edible composition obtained by the method of any one of claims 56 to 109.

111. A method for preparing an edible composition, the method comprising the steps of:

Processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises a flocculant that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein;

An edible composition comprising the protein product is formulated.

112. A method for preparing an edible composition, the method comprising the steps of:

Processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises an adsorbent that is reversibly attached to one or more components of the more than one recombinant cell by-product and that is substantially unattached to the recombinant protein;

An edible composition comprising the protein product is formulated.

113. A method for preparing an edible composition, the method comprising the steps of:

processing the composition under conditions that separate the recombinant protein from the more than one recombinant cell by-product, wherein the processing step comprises digesting the recombinant protein or an enzyme that digests the EPS;

An edible composition comprising the protein product is formulated.