A method of producing a kumamolisin-as polypeptide

Abstract

The present disclosure is directed to methods of producing Kuma polypeptides. The method of producing Kuma polypeptides can comprise heat treatment and purification by calcium precipitation. The present disclosure is also directed to methods of producing Kuma polypeptides, wherein the Kuma polypeptide comprises a polypeptide of Kuma062.

Description

A METHOD OF PRODUCING A KUMAMOLISIN-AS POLYPEPTIDEREFERENCE TO RELATED APPLICATIONS

[0001] This application claims the priority benefit of U.S. Provisional Application No. 63 / 586,317, filed on September 28, 2023, and U.S. Provisional Application No. 63 / 673,262, filed on July 19, 2024, each of which is incorporated herein by reference in its entirety.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The contents of the sequence listing text file named“3817_217PC02_Sequencelisting_ST26”, which was created on September 26, 2024 and is 33,672 bytes in size, is hereby incorporated by reference in its entirety.FIELD OF DISCLOSURE

[0003] The present disclosure relates to methods for producing Kumamolisin-As polypeptides.BACKGROUND

[0004] Kumamolisin-As (Kuma) enzymes from Alicyclobacillus sendaiensis have been identified as strong therapeutic agents for treating Celiac disease. These enzymes enhance degradation of the Proline-Glutamine-rich peptides in gluten, resulting in a potentially revolutionary therapy for patients that cannot break down gluten on their own. However, current methods of producing Kuma enzymes are time consuming and expensive. Therefore, there is a need in the art to efficiently produce Kuma enzymes.BRIEF DESCRIPTION OF THE FIGURES

[0005] FIG. 1 depicts a representative flow diagram for production of a Kuma polypeptide.BRIEF SUMMARY

[0006] Some aspects of the present disclosure are directed to a method of producing a Kuma polypeptide comprising heat treating a cell suspension comprising one or more host cells comprising the polypeptide in a cell suspension buffer.

[0007] In some aspects, the cell suspension buffer comprises a divalent cation. In some aspects, the divalent cation is Ca2+, Mg2+, Be2+, Mn2+, Zn2+, Sr2+, Ba2+, Cu2+, or Fe2+, or any combination thereof. In some aspects, the divalent cation is a calcium salt. In some aspects, the cell suspension buffer comprises from about 1 mmol / L CaCh to about 10 mmol / L CaCh. In some aspects, the cell suspension buffer comprises about 3.4 mmol / L CaCh.

[0008] In some aspects, the heat treatment comprises heating the cell suspension to about 40°C to about 90°C for about 5 seconds to about 15 minutes, or longer. In some aspects, the heat treatment comprises heating the cell suspension to about 65°C for 1 minute.

[0009] In some aspects, the heat treatment further comprises a cooling step. In some aspects, the cooling step comprises cooling the cell suspension to about 0°C, to about 5°C, to about 10°C, or to about 15°C. In some aspects, the cooling step comprises cooling the cell suspension to about 10°C.

[0010] In some aspects, the cell suspension buffer has a pH of from about 7.2 to about 10.2. In some aspects, the cell suspension buffer has a pH of about 9.2.

[0011] In some aspects, the cell suspension buffer comprises arginine. In some aspects, the cell suspension buffer comprises about 4.2 g / L arginine.

[0012] In some aspects, the host cells are lysed into a lysed cell mixture as a result of the heat treatment.

[0013] In some aspects, the method further comprises subjecting the lysed cell mixture to a flocculation.

[0014] In some aspects, the method further comprises purifying the lysed cell mixture by calcium precipitation, thereby generating a purification mixture. In some aspects, the calcium precipitation comprises adding a phosphate solution to the purification mixture to result in a final phosphate concentration of about 10 mmol / L to about 50 mmol / L. In some aspects, the final phosphate concentration of the purification mixture is about 35 mmol / L. In some aspects, the calcium precipitation comprises adding (i) calcium acetate or (ii) a mix of calcium acetate and calcium hydroxide to the purification mixture to resultin a final concentration of calcium salt from about 10 mmol / L to about 90 mmol / L. In some aspects, the final concentration of calcium salt in the purification mixture is about 70 mmol / L.

[0015] In some aspects, the purification mixture has a pH of about 6.5 to about 9.10016] In some aspects, the calcium precipitation is conducted at a temperature of from about 0 °C to about 10 °C. In some aspects, the calcium precipitation is conducted at about 10 °C.

[0017] In some aspects, the Kuma polypeptide comprises Kuma062 or a variant thereof. In some aspects, the Kuma polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0018] In some aspects, the method further comprises centrifugation, filtration, ultrafiltration / diafiltration, formulation, sterile filtration, spray drying, packing, storage, changing the arginine buffer, concentrating the polypeptide, or any combination thereof.

[0019] In some aspects, the arginine buffer is changed to a formulation buffer selected from the group consisting of about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, and about 70 mM NaCl; about 25 mM Glycine and about 70 mM NaCl; about 27 mM sodium acetate (Na Acetate), about 3 mM Taurin, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Glycylglycine, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM sodium citrate, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; about 20 mM K2HPO4 and about 20 mM Glycine; about 20 mM K2HPO4, about 20 mM Glycine, and about 70 mM NaCl; about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, about 25 mM Glycine, and about 70 mM NaCl; about 28.04 mM K2HPO4, about 1.96 mM KH2PO4, about 30 mM Glycine, and about 70 mM NaCl; about 30 mM Glycine and about 70 mM NaCl; about 50 mM Glycine and about 70 mM NaCl; about 45 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; 25 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 25 mM K2HPO4, about lOmM Glycine, and about 70 mM NaCl; about 25 mM Na2HPC>4, about 5mM Glycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 13 mM Tris, about 0.05% polysorbate 80, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; and about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.

[0020] In some aspects, the formulation buffer further comprises from about 20% to about 50% trehalose. In some aspects, the formulation buffer further comprises about 25% trehalose. In some aspects, the formulation buffer further comprises about 47% trehalose.

[0021] In some aspects, the concentration of the Kuma polypeptide is from about 1 g / L to about 150 g / L.

[0022] In some aspects, the one or more processing steps comprises spray drying. In some aspects, the spray-drying solution comprises silica. In some aspects, the spraydrying solution does not comprise silica.

[0023] Some aspects of the present disclosure are directed to a composition comprising a Kuma polypeptide and a formulation buffer. In some aspects, the formulation buffer is selected from the group consisting of about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, and about 70 mM NaCl; about 25 mM Glycine and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Taurin, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Glycylglycine, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM sodium citrate, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; about 20 mM K2HPO4 and about 20 mM Glycine; about 20 mM K2HPO4, about 20 mM Glycine, and about 70 mM NaCl; about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, about 25 mM Glycine, and about 70 mM NaCl; about 28.04 mM K2HPO4, about 1.96 mM KH2PO4, about 30 mM Glycine, and about 70 mM NaCl; about 30 mM Glycine and about 70 mM NaCl; about 50 mM Glycine and about 70 mM NaCl; about 45 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; 25 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 25 mM K2HPO4, about lOmM Glycine, and about 70 mM NaCl; about 25 mM Na2HPC>4, about 5mM Glycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 13 mM Tris, about 0.05% polysorbate 80, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; and about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.

[0024] In some aspects, the formulation buffer does not comprise trehalose. In some aspects, the formulation buffer further comprises from about 20% to about 50% trehalose. In some aspects, the formulation buffer further comprises about 25% trehalose. In some aspects, the formulation buffer further comprises about 47% trehalose.

[0025] In some aspects, the composition further comprises silica. In some aspects, the composition does not comprise silica.

[0026] In some aspects, the concentration of the Kuma polypeptide is from about 1 g / L to about 150 g / L. In some aspects, the concentration of the Kuma polypeptide is about 100 g / L. In some aspects, the concentration of the Kuma polypeptide is about 2 g / L.

[0027] Some aspects of the present disclosure are directed to a process for producing a dry powder drug substance comprising a Kuma polypeptide comprising suspending one or more host cells comprising the Kuma polypeptide in a cell suspension buffer; heat treating the cell suspension; flocculating the cell suspension; purifying the Kuma polypeptide by calcium precipitation; concentrating the Kuma polypeptide in a solution; filtering the concentrated solution in a formulation buffer, thereby producing a spraydrying solution comprising the Kuma polypeptide solution in the formuation buffer; and spray-drying the spray-drying solution comprising the Kuma polypeptide. Some aspects of the present disclosure are directed to the dry powder drug substance comprising the Kuma polypeptide prepared using the process described herein.

[0028] In some aspects, the cell suspension buffer comprises Ca2+.

[0029] In some aspects, the heat treatment comprises heating the cell suspension at about65°C for 1 minute. In some aspects, the heat treatment further comprises a cooling step, wherein the cooling step comprises cooling the cell suspension to about 0°C, to about 5°C, to about 10°C, or to about 15°C.

[0030] In some aspects, the cell suspension has a pH of about 9.4.

[0031] In some aspects, the phosphate-glycine buffer comprises 12.5 mM K2HPO4, 12.5 mM Na2HPC>4, 10 mM Glycine, and 70 mM NaCl.

[0032] In some aspects, the formulation buffer further comprises 25% or 47% trehalose.

[0033] In some aspects, the formulation buffer further comprises silica. In some aspects, the formulation buffer does not comprise silica.

[0034] In some aspects, the Kuma polypeptide comprises Kuma062 or a variant thereof. In some aspects, the Kuma polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

[0035] Some aspects of the present disclosure are directed to a composition comprising a Kuma polypeptide and a formulation buffer comprising 12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, and 70 mM NaCl.

[0036] In some aspects, the composition further comprises 25% trehalose. In some aspects, the composition further comprises 47% trehalose.

[0037] In some aspects, the composition further comprises silica.

[0038] In some aspects, the Kuma polypeptide comprises Kuma062 or a variant thereof.In some aspects, the Kuma polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.DETAILED DESCRIPTION

[0039] The present disclosure provides methods of producing a Kuma polypeptide wherein the method comprises a heat treatment.

[0040] Some aspects of the present disclosure are directed to a method of producing a Kuma polypeptide comprising heat treating a cell suspension comprising one or more host cells comprising the polypeptide. In some aspects, the method further provides purifying the Kuma polypeptide by calcium precipitation.

[0041] Before the present disclosure is described in greater detail, it is to be understood that this disclosure is not limited to the particular compositions or process steps described, which, of course, vary. As will be apparent to those of skill in the art upon reading this disclosure, each of the individual aspects described and illustrated herein has discrete components and features which can be readily separated from or combined with the features of any of the other several aspects without departing from the scope or spirit of the present disclosure. Any recited method can be carried out in the order of events recited or in any other order that is logically possible.

[0042] The headings provided herein are not limitations of the various aspects of the disclosure, which can be defined by reference to the specification as a whole. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting.Definitions

[0043] In order that the present disclosure can be more readily understood, certain terms are first defined. As used in this application, except as otherwise expressly provided herein, each of the following terms shall have the meaning set forth below. Additional definitions are set forth throughout the application.

[0044] As used herein, the singular forms "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. "And" as used herein is interchangeably used with "or" unless expressly stated otherwise. The terms "comprising, "having," "including," and "containing" are to be construed as open-ended terms (z.e., meaning "including, but not limited to") unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value recited or falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited.

[0045] Furthermore, "and / or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term "and / or" as used in a phrase such as "A and / or B" herein is intended to include "A and B," "A or B," "A" (alone), and "B" (alone). Likewise, the term "and / or" as used in a phrase such as "A, B, and / or C" is intended to encompass each of the following aspects: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone). In addition, "or" is used mean an open list of the components in the list. For example, "wherein X comprises A or B" means X comprises A, X comprises B, X comprises A and B, or X comprises A or B and any other components.

[0046] The term "about" or "approximately" usually means within 10%, within 5%, or within 1%, of a given value or range.]0047[ The term "amino acid" refers to the twenty common naturally occurring amino acids. Naturally occurring amino acids include: alanine (Ala; A), asparagine (Asn; N), aspartic acid (Asp; D), arginine (Arg; R), cysteine (Cys; C), glutamic acid (Glu; E), glutamine (Gin; Q), glycine (Gly; G), histidine (His; H), isoleucine (He; I), leucine (Leu; L), lysine (Lys; K), methionine (Met; M), phenylalanine (Phe; F), proline (Pro; P), serine (Ser; S), threonine (Thr; T), tryptophan (Trp; W), tyrosine (Tyr; Y), and valine (Vai; V).

[0048] As used herein, a “calcium salt” refers to a compound containing calcium ions (Ca2+) as cations. Examples of calcium salts can include, but are not limited to, calcium acetate, calcium carbonate, calcium chloride, calcium citrate, calcium gluconate, calcium lactate, calcium nitrate, calcium phosphate, calcium sulfate, or any combination thereof.

[0049] As used herein, a "conservative amino acid substitution" is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties (e.g., charge or hydrophobicity). In general, aconservative amino acid substitution will not substantially change the functional properties of a polypeptide. In cases where two or more amino acid sequences differ from each other by conservative substitutions, the percent sequence identity or degree of similarity may be adjusted upwards to correct for the conservative nature of the substitution. Means for making this adjustment are well-known to those of skill in the art. See, e.g., Pearson (1994) Methods Mol. Biol. 24: 307-331, herein incorporated by reference. Examples of groups of amino acids that have side chains with similar chemical properties include (1) aliphatic side chains: glycine, alanine, valine, leucine and isoleucine; (2) aliphatic-hydroxyl side chains: serine and threonine; (3) amide-containing side chains: asparagine and glutamine; (4) aromatic side chains: phenylalanine, tyrosine, and tryptophan; (5) basic side chains: lysine, arginine, and histidine; (6) acidic side chains: aspartate and glutamate, and (7) sulfur-containing side chains are cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine- isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, glutamate-aspartate, and asparagine-glutamine. Alternatively, a conservative replacement is any change having a positive value in the PAM250 log-likelihood matrix disclosed in Gonnet et al. (1992) Science 256: 1443-1445, herein incorporated by reference. A "moderately conservative" replacement is any change having a nonnegative value in the PAM250 loglikelihood matrix.[00501 As used herein, the term "divalent cation" refers to a positively charged ion having valence of 2+ Examples of divalent cation include, but are not limited to, Ca2+, Mg2+, Be2+, Mn2+, Zn2+, Sr2+, Ba2+, Cu2+, and Fe2+.|0051| As used herein, the term “heat treatment,” “heat treating,” “heat treat,” or the like, refers to heating a sample, e.g., a cell suspension, at a temperature from about 40°C to about 90°C for about 5 seconds to about 15 minutes. A heat treatment is a gentle, yet effective method of lysing cells, as high temperatures can improve peptide solubility and increase a peptide’s emulsifying properties. In some aspects, the heat treatment further comprises a cooling step. In some aspects, the heat treatment comprises subjecting the sample, e.g., the cell suspension, to a temperature of about 40°C, about 45°C, about 50°C, about 55°C, about 60°C, about 65°C, about 70°C, about 75°C, about 80°C, about 85°C, or about 90°C. In some aspects, the sample, e.g., the cell suspension, is held at the temperature for about 5 seconds, about 10 seconds, about 15 seconds, about 20 seconds, about 25 seconds, about 30 seconds, about 40 seconds, about 50 seconds, about 60seconds, about 70 seconds, about 80 seconds, about 90 seconds, about 2 minutes, about2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 7.5 minutes, about 8 minutes, about 8.5 minutes, about 9 minutes, about 9.5 minutes, about 10 minutes, about 10.5 minutes, about 11 minutes, about 11.5 minutes, about 12 minutes, about 12.5 minutes, about 13 minutes, about 13.5 minutes, about 14 minutes, about 14.5 minutes, or about 15 minutes. In some aspects, the heat treatment comprises heating a sample, e.g., a cell suspension, at 65 °C for about 1 minute. In some aspects, the heat treatment comprises heating the cell suspension from about 15°C or below to about between 60°C and 70°C (e.g., about 65°C) within from about 1 to about 3 minutes (e.g., within about 1 minute, within about 1.5 minutes, within about 2 minutes, within about 2.5 minutes, within about 3 minutes), cooling the cell suspension or purification mixture in a passive cooling step to between about 40°C to 52°C (e.g., about 40°C, about 42°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 48.5°C, about 49°C, about 49.5°C, about 50°C, about 50.5°C, about 51°C, about 51.5°C, or about 52°C) within between about 1 and about 7 minutes (e.g., within about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, or about 7 minutes), and further cooling the cell suspension or purification mixture in an active cooling step to between about 0°C and about 20°C (e.g., about 0°C, about 0.5°C, about 1°C, about 1.5°C, about 2°C, about 2.5°C, about 3°C, about 3.5°C, about 4°C, about 4.5°C, about 5°C, about 5.5°C, about 6°C, about 6.5°C, about 7°C, about 7.5°C, about 8°C, about 8.5°C, about 9°C, about 9.5°C, about 10°C, about 10.5°C, about 11°C, about 11.5°C, about 12°C, about 12.5°C, about 13°C, about 13.5°C, about 14°C, about 14.5°C, about 15°C, about 15.5°C, about 16°C, about 16.5°C, about 17°C, about 17.5°C, about 18°C, about 18.5°C, about 19°C, about 19.5°C, or about 20°C) within from about 30 seconds to about 10 minutes (e.g., about 30 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 7.5 minutes, about 8 minutes, about8.5 minutes, about 9 minutes, about 9.5 minutes, or about 10 minutes). In some aspects, the heat treatment comprises cooling the cell suspension or purification mixture to a temperature between about 1.5 °C and about 5 °C. As used herein, the term “passivecooling,” “passively cool,” or the like, refers to cooling a mixture, e.g., a cell suspension or a mixture comprising the lysed cells, without applying an external cooling mechanism, e.g., heat exchange, to the mixture. In an unlimiting example, by passive cooling, a mixture is placed under an ambient temperature, e.g., room temperature, to cool down. As used herein, the term “active cooling,” “actively cool,” or the like, refers to cooling a mixture, e.g., a cell suspension or a mixture comprising lysed cells by applying an external cooling mechanism, e.g., heat exchange, to the mixture. In some aspects, active cooling is achieved by applying heat exchange to the mixture to be cooled.

[0052] In some aspects, the heat treatment comprises heating the cell suspension from about 15°C or below to about 65°C within about 1 minute or about 1.5 minutes, passively cooling the cell suspension or purification mixture to about 50°C within about between 2 minutes and 5 minutes, and actively cooling the cell suspension or purification mixture to about 10°C within about 1 minute. In some aspects, the temperature referred to in a heat treatment is the temperature of the cell suspension or purification mixture. In some aspects, the heat treatment comprises raising the temperature of the cell suspension or purification mixture.

[0053] As used herein, the term “Kumamolisin-As” or “Kuma,” refers to enzymes from Alicyclobacillus sendaiensis that degrade the Proline-Glutamine-rich peptides in gluten. In some aspects, a Kuma polypeptide is a Kuma polypeptide disclosed in US20180142226 Al , US20160194621 , US20140178355 Al , and / or WO 2022 / 094177, which are each incorporated by reference in their entireties.

[0054] As used herein, the term “Kuma062,” refers to a polypeptide having an amino acid sequence as shown below: SDMEKPWKEGEEARAVLQGHARAQAPQAVDKGPVAGDERMAVTVVLRRQRA GELAAHVERQAAIAPHAREHLKREAFAASHGASLDDFAELRRFADAHGLALDRA NVAAGTAVLSGPDDAINRAFGVELRHFDHPDGSYRSYLGEVTVPASIAPMIEAVL GLDTRPVARRRFRMQRRAEGGFEARSQAAAPTAYTPLDVAQAYQFPEGLDGQG QCIAIIELGGGYDEASLAQYFASLGVPAPQVVSVSVDGASNQPTGDPEGPDGEVT LDIEVAGALAPGAKFAVYFAPDTTAGFLDAITTAIHDPTLKPSVVSISWSMPEDS WTSAAIAAMNRAFLDAAALGVTVLAAAGDQGSTSGEQDGLYHVHFPAASPYVL ACGGTRLVASGGRIAQETVWNQGPDGGATGGGVSRIFPLPAWQEHANVPPSANP GASSGRGVPDLAGNADPQTGYEVVIDGEATVTGGTSAVAPLFAALVARINQKLGKAVGYLNPTLYQLPADVFHDITEGNNDIANRAQIYQAGPGWDPCTGLGSPIGVRL LQALLPSASQPQP (SEQ ID NO: 1).Kuma062 is a Kuma polypeptide.

[0055] The term "mutation," as used herein, refers to insertion, deletion, or substitution of one or more amino acids in a polypeptide or of one or more nucleotides in a polynucleotide, as compared to a control or reference (z.e., an unmodified polypeptide).

[0056] As used herein, the term "polypeptide" is used in its broadest sense to refer to a sequence of subunit amino acids, whether naturally occurring or of synthetic origin. The polypeptides of the disclosure may comprise L-amino acids, D-amino acids (which are resistant to L-amino acid-specific proteases in vivo), or a combination of D- and L-amino acids. The polypeptides may be linked to other compounds to promote an increased halflife in vivo, such as by PEGylation, HESylation, PSAylation, or glycosylation. Such linkage can be covalent or non-covalent as is understood by those of skill in the art. In some aspects, the polypeptides are linked to any other suitable linkers, including but not limited to any linkers that can be used for purification or detection (such as FLAG or His tags).

[0057] The term “variant,” as used herein, refers to a polypeptide that comprises one or more amino acid insertions, substitutions, or deletions relative to a reference polypeptide. In certain aspects, a variant polypeptide has at least about 75% amino acid identity, e.g., at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or about 100% sequence identity, to a reference polypeptide sequence. In some aspects, a variant of a reference polypeptide maintains one or more functions, activities, and / or structures of the reference polypeptide or polynucleotide. For example, a variant of a Kuma062 disclosed herein maintains the function to degrade gluten and / or gliadin effectively. In another example, a variant of a polypeptide encoding a Kuma062 encodes a functional Kuma062.

[0058] In some aspects, a variant of Kuma062 is a Kuma polypeptide disclosed in US20180142226A1, US20140178355A1, and / or WO 2022 / 094177, which are each incorporated by reference in their entireties.

[0059] In some aspects, a variant of a Kuma062 comprises:

[0060] Ser at residue 466, Glu at residue 266, and Asp at residue 270; and[00611 Glu at residue 261, Thr at residue 268, Gin at residue 353, Ser at residue 357, Gin at residue 398, Gin at residue 448, Thr at residue 462.

[0062] In some aspects, a variant of a Kuma062 comprises an amino acid sequence as set forth in SEQ ID No: 22.100631 In some aspects, the amino acid sequence as set forth in SEQ ID NO: 22 can comprise an unspecified amino acid (“Xaa” or “X”) at any one of amino acid positions 220, 261, 267, 268, 269, 318, 319, 353, 357, 367, 398, 401, 405, 423, 448, 460, and 462. In some aspects, the amino acid sequence as set forth in SEQ ID NO: 22 can further comprise an unspecified amino acid (“Xaa” or “X”) at any one of amino acid positions selected from the group consisting of 104, 170, 171, 172, 173, and / or 455.

[0064] In some aspects, a variant of a Kuma062 comprises an amino acid sequence as set forth in SEQ ID No: 22 can comprise an amino acid sequence at least 75% identical to the amino acid sequence of SEQ ID NO: 1, wherein

[0065] Ser at residue 466, Glu at residue 266, and Asp at residue 270; and

[0066] the polypeptide can comprise an amino acid at one or more Xaa residues selected from the group consisting of 220D / N / Q / H, 26 IE, 267S / T / A, 268L / T, 269A / T / V, 318A, 319M, 353A / E / Q / R / Y, 357N / S / Q / T, 367F / Q, 398Q, 401 S / Q, 405S, 423K, 448E / N / Q, 460R, and 462A / L / M / Q / R / T / V.

[0067] In some aspects, the polypeptide further comprises an amino acid at one or more Xaa amino acid positions selected from the group consisting of 104H; 170R / A / S; 171R / A / S; 172R / S, 173S, and / or 455V.(0068] The position number of an amino acid residue in a variant of a reference polypeptide, e.g., Kuma062, refers to the position number of the corresponding amino acid residue in the reference polypeptide, e.g., Kuma062. Using Kuma062 as a reference polypeptide for illustration, to assign a position number of an amino acid residue in a variant of Kuma062, the sequence of the variant of Kuma062 is aligned with Kuma062 (SEQ ID NO: 1) using one of any sequence analysis software described herein. The position number of the amino acid residue on the variant is the position number of the corresponding amino acid on Kuma062 that is aligned to the amino acid residue on the variant in the alignment of the two sequences. If there is one or more insertions in the variant of Kuma062, the position number of one or more amino acids in the insertion is designated as n.m, wherein “n” is the position number of the amino acid residue immediately preceding the insertion and “m” is the number of the inserted amino acidresidue counting from the immediately preceding amino acid residue. For example, in an alignment of a hypothetical variant of Kuma062 with the first 20 amino acid residues of Kuma062 as shown below,Kuma062 SD - MEKPWKEGEEARAVLQGHKuma062 variant (hypothetical) SDAGLMEKPWKEGEEARAVLQGH three amino acids (“AGL”) were inserted after position 2 (“D”). The position number of these three amino acid residues are designated as 2.1 for A, 2.2 for G, and 2.3 for L, respectively.

[0069] Sequence identity is typically measured using sequence analysis software. Protein analysis software matches similar sequences using measures of similarity assigned to various substitutions, deletions, and other modifications, including conservative amino acid substitutions. For instance, GCG software contains programs such as Gap and Bestfit, which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild type protein and a mutein thereof. See, e.g., GCG Version 6.1. Polypeptide sequences also can be compared using FASTA using default or recommended parameters, a program in GCG Version 6.1. FASTA (e.g., FASTA2 and FASTA3) provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences (Pearson (2000) supra).Another non-limiting example of algorithm that can be used to compare a sequence of the disclosure to a database containing a large number of sequences from different organisms is the computer program BLAST, e.g., BLASTP or TBLASTN, using default parameters. See, e.g., Altschul et al. (1990) J. Mol. Biol. 215:403-410 and Altschul et al. (1997) Nucleic Acids Res. 25:3389-402, each of which is incorporated by reference herein in its entirety.

[0070] Various aspects described herein are described in further detail in the following subsections.Methods and Compositions of the Disclosure(0071 [ Some aspects of the present disclosure provide methods of producing a Kuma polypeptide. In some aspects, the method comprises a heat treatment.

[0072] Some aspects of the present disclosure provide methods of producing a Kuma polypeptide comprising heat treating a cell suspension comprising one or more host cellscomprising a Kuma polypeptide in a cell suspension buffer. In some aspects, the method further comprises suspending one or more host cells comprising the polypeptide in a cell suspension buffer. In some aspects, the method further comprises purifying the Kuma polypeptide by calcium precipitation.100731 Any host cell can be utilized in accordance with the methods disclosed herein. In some aspects, the host cells comprise prokaryotic cells. In some aspects, the host cells comprise eukaryotic cells. In some aspects, the cells are transiently transfected or transduced. In some aspects, the cells are stably transfected or transduced. Such transfection and transduction of expression vectors into prokaryotic and eukaryotic cells can be accomplished via any technique, including but not limited to standard bacterial transformations, calcium phosphate co-precipitation, electroporation, or liposome mediated-, DEAE dextran mediated-, poly cationic mediated-, or viral mediated transfection. (See, e.g., Molecular Cloning: A Laboratory Manual (Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press; Culture of Animal Cells: A Manual of Basic Technique, 2ndEd. (R.I. Freshney. 1987. Liss, Inc. New York, NY). One skilled in the art will appreciate that host cells can have different nutrition requirements and / or might require different culture conditions for optimal growth and polypeptide expression, and will be able to modify conditions as needed. In some aspects, the host cells comprise E. coli cells.

[0074] In some aspects, fermentation technology is applied in the methods disclosed herein to increase cell density and / or protein productivity. Fermentation provides control over key chemical, physical and biological parameters that affect cell growth, as well as recombinant polypeptide production. These parameters can include, but are not limited to, temperature, dissolved oxygen (DO) level, pH, nutrient supply, or any combination thereof.

[0075] In some aspects, the host cell medium comprises a divalent cation, e.g., calcium salt. In some aspects, the divalent cation is an exogenous divalent cation added into the host cell medium. Without wishing to be bound by any theory, it is hypothesized that a divalent cation, e.g., calcium salt may help to stabilize the Kuma polypeptide, e.g., to maintain the enzymatic activity of the Kuma polypeptide. In some aspects, the host cell medium comprises a calcium salt. In some aspects, the host cell medium comprises CaCh. In some aspects, the amount of CaCh in the host cell medium can be between about 0 mmol / L to about 10 mmol / L (e.g., ranging from about 0 mmol / L to about 9mmol / L, 1 mmol / L to about 9 mmol / L, from about 2 mmol / L to about 9 mmol / L, from about 3 mmol / L to about 9 mmol / L, from about 4 mmol / L to about 9 mmol / L, from about 5 mmol / L to about 9 mmol / L, from about 6 mmol / L to about 9 mmol / L, from about 7 mmol / L to about 9 mmol / L, from about 8 mmol / L to about 9 mmol / L, from about 1 mmol / L to about 8 mmol / L, from about 1 mmol / L to about 7 mmol / L, from about 1 mmol / L to about 6 mmol / L, from about 1 mmol / L to about 5 mmol / L, from about 1 mmol / L to about 4 mmol / L, from about 1 mmol / L to about 3 mmol / L, or from about 1 mmol / L to about 2 mmol / L). In some aspects, the host cell medium comprises about 0 mmol / L CaCh, about 1 mmol / L CaCh, about 1.5 mmol / L CaCh, about 2 mmol / L CaCh, about 2.5 mmol / L CaCh, about 3 mmol / L CaCh, about 3.5 mmol / L CaCh, about 4 mmol / L CaCh, about 4.5 mmol / L CaCh, about 5 mmol / L CaCh, about 5.5 mmol / L CaCh, about 6 mmol / L CaCh, about 6.5 mmol / L CaCh, about 7 mmol / L CaCh, about 7.5 mmol / L CaCh, about 8 mmol / L CaCh, about 8.5 mmol / L CaCh, about 9 mmol / L CaCh, about 9.5 mmol / L CaCh, or about 10 mmol / L CaCh. In some aspects, the amount of CaCh in the host cell medium is about 3.4 mmol / L. In some aspects, the host cell medium does not comprise exogenous CaCh.

[0076] In some aspects, the one of more host cells are washed after fermentation. In some aspects, the cell wash dilutes the wet cell weight (WCW). In some aspects, the cell wash comprises dilution with an Arginine buffer (Arg-buffer) comprising a calcium salt. In some aspects, the cells are resuspended. In some aspects, the cell suspension buffer comprises an Arg-buffer comprising a divalent cation, e.g., calcium salt. In some aspects, the Arg-buffer used in the cell wash has a similar, substantially the same, or the same composition to the cell suspension buffer. For example, the Arg-buffer used in the cell wash may comprise Arginine and / or calcium salt with a similar or identical concentration to the cell suspension buffer. The cell suspension buffer may or may not comprise additional components, such as another salt. In some aspects, the divalent cation is Ca2+, Mg2+, Be2+, Mn2+, Zn2+, Sr2+, Ba2+, Cu2+, or Fe2+, or any combination thereof. In some aspects, the divalent cation is a calcium salt. In some aspects, the cell suspension buffer comprises from about 1 mmol / L CaCh to about 10 mmol / L CaCh (e.g., from about 1 mmol / L to about 9 mmol / L, from about 2 mmol / L to about 9 mmol / L, from about 3 mmol / L to about 9 mmol / L, from about 4 mmol / L to about 9 mmol / L, from about 5 mmol / L to about 9 mmol / L, from about 6 mmol / L to about 9 mmol / L, from about 7 mmol / L to about 9 mmol / L, from about 8 mmol / L to about 9 mmol / L, from about 1mmol / L to about 8 mmol / L, from about 1 mmol / L to about 7 mmol / L, from about 1 mmol / L to about 6 mmol / L, from about 1 mmol / L to about 5 mmol / L, from about 1 mmol / L to about 4 mmol / L, from about 1 mmol / L to about 3 mmol / L, or from about 1 mmol / L to about 2 mmol / L). In some aspects, the cell suspension buffer comprises about 1 mmol / L CaCh, about 1.5 mmol / L CaCh, about 2 mmol / L CaCh, about 2.5 mmol / L CaCh, about 3 mmol / L CaCh, about 3.5 mmol / L CaCh, about 4 mmol / L CaCh, about4.5 mmol / L CaCh, about 5 mmol / L CaCh, about 5.5 mmol / L CaCh, about 6 mmol / L CaCh, about 6.5 mmol / L CaCh, about 7 mmol / L CaCh, about 7.5 mmol / L CaCh, about 8 mmol / L CaCh, about 8.5 mmol / L CaCh, about 9 mmol / L CaCh, about 9.5 mmol / L CaCh, or about 10 mmol / L CaCh. In some aspects, the cell suspension buffer comprises about 3.4 mmol / L CaCh.

[0077] In some aspects, the Arg-buffer or the cell suspension buffer comprises from about 0.2 g / L NaCl to about 0.9 g / L NaCl (e.g., from about 0.2 g / L to about 0.8 g / L, from about 0.3 g / L to about 0.7 g / L, or from about 0.4 g / L to about 0.6 g / L). In some aspects, the Arg-buffer comprises about 0.2 g / L NaCl, about 0.3 g / L NaCl, about 0.4 g / L NaCl, about 0.45 g / L NaCl, about 0.5 g / L NaCl, about 0.55 g / L NaCl, about 0.6 g / L NaCl, about 0.65 g / L NaCl, about 0.7 g / L NaCl, about 0.75 g / L NaCl, about 0.8 g / L NaCl. In some aspects, the Arg-buffer or the cell suspension buffer comprises about 0.58 g / L NaCl.100781 In some aspects, the Arg-buffer or the cell suspension buffer comprises from about 1 g / L L- Arginine HC1 to about 15 g / L L-Arginine HC1 (e.g., from about 2 g / L L- Arginine HC1 to about 6 g / L L-Arginine HC1, from about 3 g / L L-Arginine HC1 to about 5 g / L L-Arginine HC1, or about 4 g / L L-Arginine HC1). In some aspects, the Arg-buffer comprises about 1 g / L L-Arginine HC1, about 2 g / L L-Arginine HC1, about 3 g / L L- Arginine HC1, about 3.5 g / L L-Arginine HC1, about 4 g / L L-Arginine HC1, about 4.1 g / L L-Arginine HC1, about 4.2 g / L L-Arginine HC1, about 4.3 g / L L-Arginine HC1, about 4.4 g / L L-Arginine HC1, about 4.5 g / L L-Arginine HC1, about 5 g / L L-Arginine HC1, about5.5 g / L L-Arginine HC1, about 6 g / L L-Arginine HC1, about 7 g / L L-Arginine HC1, about7.5 g / L L-Arginine HC1, about 8 g / L L-Arginine HC1, about 8.5 g / L L-Arginine HC1, about 9 g / L L-Arginine HC1, about 9.5 g / L L-Arginine HC1, about 10 g / L L-Arginine HC1, about 10.5 g / L L-Arginine HC1, about 11 g / L L-Arginine HC1, about 11.5 g / L L- Arginine HC1, about 12 g / L L-Arginine HC1, about 12.5 g / L L-Arginine HC1, about 13 g / L L-Arginine HC1, about 13.5 g / L L-Arginine HC1, about 14 g / L L-Arginine HC1, about14.5 g / L L-Arginine HC1, or about 15 g / L L-Arginine HC1.

[0079] In some aspects, the Arg-buffer or the cell suspension buffer has a pH from about 7.2 to about 10.2. In some aspects, the cell suspension has a pH of about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, or about 10.2 during the wash. In some aspects, the cell suspension has a pH of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10.0 during the wash. In particular aspects, the cell suspension has a pH of about 9.2 during the wash.

[0080] In some aspects, the cells are lysed after the fermentation. In some aspects, the cells are lysed after the fermentation and after the wash. Any method for physically lysing cells to extract peptides can be used in the methods disclosed herein. In some aspects, the lysing comprises manual disruption, mechanical disruption, liquid homogenization, high frequency sound waves (sonication), temperature treatments (e.g., freeze-thaw and / or heat treatments), or any combination thereof.

[0081] In some aspects, the lysing comprises a heat treatment. In some aspects, the heat treatment comprises heating the cell suspension to about 40°C to about 90°C (e.g., to about 40°C to about 80°C, to about 40°C to about 70°C, to about 40°C to about 60°C, to about 40°C to about 50°C, to about 50°C to about 90°C, to about 50°C to about 80°C, to about 50°C to about 70°C, or to about 50°C to about 60°C) for about 5 seconds to about 15 minutes (e.g., from about 5 seconds to about 14 minutes, from about 5 seconds to about 13 minutes, from about 5 seconds to about 12 minutes, from about 5 seconds to about 11 minutes, from about 5 seconds to about 10 minutes, from about 5 seconds to about 9 minutes, from about 5 seconds to about 8 minutes, from about 5 seconds to about 7 minutes, from about 5 seconds to about 6 minutes, from about 5 seconds to about 5 minutes, from about 5 seconds to about 4.5 minutes, from about 5 seconds to about 4 minutes, from about 5 seconds to about 3.5 minutes, from about 5 seconds to about 3 minutes, from about 5 seconds to about 2.5 minutes, from about 5 seconds to about 2 minutes, from about 5 seconds to about 1.5 minutes, from about 5 seconds to about 1 minute, from about 5 seconds to about 30 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 minutes, from about 30 seconds to about 15 minutes, from about 1 minute to about 15 minutes, from about 1.5 minutes to about 15minutes, from about 2 minutes to about 15 minutes, from about 2.5 minutes to about 15 minutes, from about 3 minutes to about 15 minutes, from about 3.5 minutes to about 15 minutes, from about 4 minutes to about 15 minutes, from about 4.5 minutes to about 15 minutes, from about 5 minutes to about 15 minutes, from about 6 minutes to about 15 minutes, from about 7 minutes to about 15 minutes, from about 8 minutes to about 15 minutes, from about 9 minutes to about 15 minutes, from about 10 minutes to about 15 minutes, from about 11 minutes to about 15 minutes, from about 12 minutes to about 15 minutes, from about 13 minutes to about 15 minutes, or from about 14 minutes to about 15 minutes). In some aspects, the heat treatment comprises heating the cell suspension toabout 50°C to about 80°C for about 5 seconds to about 3 minutes. In some aspects, the heat treatment comprises heating the cell suspension to about 60°C to about 70°C for about 1 minute to about 2 minutes. In some aspects, the heat treatment comprises heating the cell suspension to about 60°C to about 70°C for about 1 minute. In particular aspects, the heat treatment comprises heating the cell suspension to about 65°C for about 1 minute. In some aspects, the heat treatment comprises heating the cell suspension to about 15°C or below to about between 60°C and 70°C (e.g., about 65°C) within between about 1 minute to about 3 minutes (e.g., within about 1 minute, within about 1.5 minutes, within about 2 minutes, within about 2.5 minutes, or within about 3 minutes).100821 In some aspects, the heat treatment further comprises a cooling step. In some aspects, the cooling step comprises passively cooling the cell suspension or purification mixture to a temperature of between about 40°C and 52°C (e.g., between about 40°C and 51°C, between about 40°C and 50°C, between about 40°C and 49°C, between about 40°C and 48°C, between about 40°C and 47°C, between about 40°C and 46°C, between about 40°C and 45°C, between about 40°C and 44°C, between about 40°C and 43°C, between about 40°C and 42°C, between about 40°C and 41 °C, between about 41 °C and 52°C, between about 42°C and 52°C, between about 43 °C and 52°C, between about 44°C and 52°C, between about 45°C and 52°C, between about 46°C and 52°C, between about 47°C and 52°C, between about 48°C and 52°C, between about 49°C and 52°C, between about 50°C and 52°C, or between about 51°C and 52°C). In some aspects, the cooling step comprises passively cooling the cell suspension or purification mixture to a temperature of about 41°C, about 42°C, about 43°C, about 44°C, about 45°C, about 46°C, about 47°C, about 48°C, about 49°C, about 50°C, about 51°C, about 52°C. In some aspects, the cooling step comprises actively cooling the cell suspension or purification mixture to atemperature of about 50°C. In some aspects, the passive cooling step between about 30 seconds to about 4 minutes (e.g., about 30 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, or about 4 minutes).100831 In some aspects the cooling step comprises actively cooling the cell suspension or purification mixture to about 0°C to about 20°C (e.g., from about 0°C to about 19°C, from about 0°C to about 18°C, from about 0°C to about 17°C, from about 0°C to about 16°C, from about 0°C to about 15°C, from about 0°C to about 14°C, from about 0°C to about 13 °C, from about 0°C to about 12°C, from about 0°C to about 11°C, from about 0°C to about 10°C, from about 0°C to about 9°C, from about 0°C to about 8°C, from about 0°C to about 7°C, from about 0°C to about 6°C, from about 0°C to about 5°C, from about 0°C to about 4°C, from about 0°C to about 3 °C, from about 0°C to about 2°C, from about 0°C to about 1°C, from about 1°C to about 20°C, from about 2°C to about 20°C, from about 3°C to about 20°C, from about 4°C to about 20°C, from about 5°C to about 20°C, from about 6°C to about 20°C, from about 7°C to about 20°C, from about 8°C to about 20°C, from about 9°C to about 20°C, from about 10°C to about 20°C, from about 11 °C to about 20°C, from about 12°C to about 20°C, from about 13 °C to about 20°C, from about 14°C to about 15°C, from about 15°C to about 20°C, from about 16°C to about 20°C, from about 17°C to about 20°C, from about 18°C to about 20°C, or from about 19°C to about 20°C). In some aspects, the cooling step comprises actively cooling the cell suspension or purification mixture to about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C. In some aspects, the cooling step comprises actively cooling the cell suspension to about 10°C. In some aspects, the cooling step comprises actively cooling the cell suspension or purification mixture from a temperature of about 50°C to about 0°C to about 20°C within between about 30 seconds and about 10 minutes (e.g., about 30 seconds, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 5.5 minutes, about 6 minutes, about 6.5 minutes, about 7 minutes, about 7.5 minutes, about 8 minutes, about 8.5 minutes, about 9 minutes, about 9.5 minutes, or about 10 minutes).

[0084] In some aspects, the heat treatment comprises heating the cell suspension from about 15°C or below to about 65°C within about 1 minute or about 1.5 minutes, passively cooling the cell suspension or purification mixture to about 50°C within between about 2minutes and about 5 minutes, and actively cooling the cell suspension or purification mixture to about 10°C within about 1 minute. In some aspects, the temperature referred to in a heat treatment is the temperature of the cell suspension or purification mixture.

[0085] In some aspects, the heat treatment further comprises subjecting the cell suspension buffer to a pH from about 7.2 to about 10.2. In some aspects, the cell suspension has a pH of about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, or about 10.2 during the heat treatment. In some aspects, the cell suspension has a pH of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10.0 during the heat treatment. In particular aspects, the cell suspension has a pH of about 9.2 during the heat treatment.

[0086] In some aspects, the cell suspension buffer comprises arginine. In some aspects, the cell suspension buffer comprises from about 1 g / L arginine to about 15 g / L arginine (e.g., from about 1.5 g / L arginine to about 10 g / L arginine, from about 2 g / L arginine to about 5 g / L arginine, or about 4 g / L arginine). In some aspects, the cell suspension buffer comprises about 1 g / L arginine, about 2 g / L arginine, about 3 g / L arginine, about 3.5 g / L arginine, about 4 g / L arginine, about 4.1 g / L arginine, about 4.2 g / L arginine, about 4.3 g / L arginine, about 4.4 g / L arginine, about 4.5 g / L arginine, about 5 g / L arginine, about 5.5 g / L arginine, about 6 g / L arginine, about 7 g / L arginine, about 7.5 g / L arginine, about 8 g / L arginine, about 8.5 g / L arginine, about 9 g / L arginine, about 9.5 g / L arginine about 10 g / L arginine, about 10.5 g / L arginine, about 11 g / L arginine, about 11.5 g / L arginine, about 12 g / L arginine, about 12.5 g / L arginine, about 13 g / L arginine, about 13.5 g / L arginine, about 14 g / L arginine, about 14.5 g / L arginine, or about 15 g / L arginine. In some aspects, the cell suspension buffer comprises about 4.2 g / L arginine.

[0087] In some aspects, the host cells are lysed into a lysed cell mixture as a result of the heat treatment.

[0088] In some aspects, the lysed cell suspension (z.e., a purification mixture comprising the lysed cells and the recombinant expressed polypeptide) are further subjected to flocculation. As used herein, “flocculation” refers to the aggregation of unstable and small particles through surface charge neutralization. As used herein, a “purificationmixture” refers to any mixture that comprises lysed cells or any components thereof and the recombinant expressed Kuma polypeptide. A purification mixture includes the mixture resulting from the lysis of the host cells or any mixture that is partially or substantially purified (z.e., a mixture that comprises the recombinant expressed polypeptide and any other components from the lysed cells, in which the components other than the recombinant expressed polypeptide are preferentially (partially or substantially) removed by any process, e.g., flocculation). In some aspects, the flocculation comprises contacting the purification mixture with one or more artificial flocculants. In some aspects, the one or more artificial flocculants comprise aluminum- based inorganic compounds, chitosan-based cationic polymers, poly-s-lysine, or any combination thereof. In some aspects, poly-s-lysine is added to the purification mixture in order to separate impurities from the purification mixture. In some aspects, the purification mixture comprises from about 0.1 g / L poly-s-lysine to about 5 g / L poly-s- lysine (e.g., from about 0.5 g / L poly-s-lysine to about 3.5 g / L poly-s-lysine, from about 1 g / L poly-s-lysine to about 3 g / L poly-s-lysine, or about 2 g / L poly-s-lysine). In some aspects, the purification mixture comprises about 0.1 g / L poly-s-lysine, about 0.5 g / L poly-s-lysine, about 1 g / L poly-s-lysine, about 1.5 g / L poly-s-lysine, about 2 g / L poly-s- lysine, about 2.5 g / L poly-s-lysine, about 3 g / L poly-s-lysine, about 3.5 g / L poly-s-lysine, about 4 g / L poly-s-lysine, about 4.5 g / L poly-s-lysine, or about 5 g / L poly-s-lysine. In some aspects, the purification mixture comprises about 2 g / L poly-s-lysine.

[0089] In some aspects, the lysed cell mixture is further subjected to a calcium precipitation thereby generating a purification mixture. Calcium precipitation is performed by addition of phosphate and / or calcium into the purification mixture.

[0090] In some aspects, the purification mixture subjected to calcium precipitation comprises calcium. Calcium precipitation is achieved by adding phosphate to the purification mixture comprising calcium in an amount that is about equimolar to the calcium, in a molar amount that is in excess to the calcium, or in a molar amount that is less than the calcium.

[0091] In some aspects, the purification mixture subjected to calcium precipitation comprises phosphate. Calcium precipitation is achieved by adding calcium salt to the purification mixture comprising phosphate in an amount that is about equimolar to the phosphate, in a molar amount that is in excess to the calcium, or in a molar amount that is less than the phosphate.

[0092] In some aspects, the purification mixture subjected to the calcium precipitation comprises a divalent cation other than calcium, e.g., Mg2+. Calcium precipitation is achieved by adding calcium salt to the purification mixture comprising a non-calcium divalent cation in an amount that is about equimolar to the non-calcium divalent cation, in a molar amount that is in excess to the non-calcium divalent cation, or in a molar amount that is less than the non-calcium divalent cation.

[0093] In some aspects, calcium precipitation is achieved by adding solid calcium phosphate to the purification mixture. The purification mixture subject to calcium precipitation may contain calcium cation in excess or phosphate in excess. Optionally, the purification mixture may contain a buffering agent to stabilize the pH during the precipitation step. In some aspects, the purification mixture subject to calcium precipitation may have been subject to heat treatment in the present of a non-calcium divalent cation, e.g., Mg2+.

[0094] In some aspects, calcium precipitation is performed by addition of phosphate stock solution at a defined pH to the purification mixture. After mixing, a calcium salt (z.e., calcium acetate) is added in sequential additions with continuous agitation. Temperature and pH are tightly controlled parameters of the flocculation and precipitation of the present disclosure.

[0095] During the calcium precipitation, a buffering agent may be included in the purification mixture to stabilize the pH during the precipitation. The buffering agent includes, but is not limited to, arginine, histidine, Tris-HCl, HEPES.

[0096] In some aspects, purifying the Kuma polypeptide comprises calcium precipitation of the purification mixture comprising the lysed cells. In some aspects, purifying the Kuma polypeptide comprises calcium precipitation after the flocculation. In some aspects, purifying the Kuma polypeptide comprises calcium precipitation before the flocculation. Without wishing to be bound by any theory, it is hypothesized that calcium precipitation removes impurities from the purification mixture and a portion of, most of, or substantially all of the Kuma polypeptide produced during fermentation remains in the soluble portion of the purification mixture. In some aspects, the calcium precipitation comprises adding a phosphate stock solution so that the final phosphate concentration is about 10 mmol / L to about 70 mmol / L (e.g., from about 15 mmol / L to about 65 mmol / L, from about 20 mmol / L to about 60 mmol / L, from about 25 mmol / L to about 55 mmol / L, from about 30 mmol / L to about 50 mmol / L, from about 35 mmol / L to about 45 mmol / L,or about 35 mmol / L). In some aspects, the final phosphate concentration is about 10 mmol / L, about 15 mmol / L, about 20 mmol / L, about 25 mmol / L, about 30 mmol / L, about 35 mmol / L, about 40 mmol / L, about 45 mmol / L, about 50 mmol / L, about 55 mmol / L, about 60 mmol / L, about 65 mmol / L, or about 70 mmol / L. In some aspects, the final phosphate concentration is about 35 mmol / L.

[0097] In some aspects, the calcium precipitation comprises adding calcium acetate or a mix of calcium acetate and calcium hydroxide to the purification mixture to result in a final concentration of the calcium salt from about 10 mmol / L to about 140 mmol / L (e.g., from about 20 mmol / L to about 130 mmol / L, from about 30 mmol / L to about 120 mmol / L, from about 35 mmol / L to about 110 mmol / L, from about 40 mmol / L to about 100 mmol / L, from about 45 mmol / L to about 95 mmol / L, from about 50 mmol / L to about 90 mmol / L, from about 55 mmol / L to about 85 mmol / L, from about 60 mmol / L to about 80 mmol / L, from about 65 mmol / L to about 75 mmol / L, or about 70 mmol / L). In some aspects, the final concentration of calcium salt is about 10 mmol / L , about 15 mmol / L, 20 mmol / L, about 25 mmol / L, about 30 mmol / L, about 35 mmol / L, about 40 mmol / L, about 45 mmol / L, about 50 mmol / L, about 55 mmol / L, about 60 mmol / L, about 65 mmol / L, about 70 mmol / L, about 75 mmol / L, about 80 mmol / L, about 85 mmol / L, about 90 mmol / L, about 95 mmol / L, about 100 mmol / L, about 105 mmol / L, about 110 mmol / L, about 115 mmol / L, about 120 mmol / L, about 125 mmol / L, about 130 mmol / L, about 135 mmol / L, or about 140 mmol / L. In some aspects, the final concentration of calcium salt in the purification mixture is about 70 mmol / L.

[0098] In some aspects, the purification mixture has a pH of about 6.5, about 6.6, about6.7, about 6.8, about 6.9, about 7.0, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, or about 9.2 during the calcium precipitation. In some aspects, the purification mixture has a pH of about 7.2 to about 8.5 during the calcium precipitation. In some aspects, the purification mixture has a pH of about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about7.8, about 7.9, about 8, about 8.1, about 8.2, about 8.3, about 8.4, or about 8.5 during the calcium precipitation.

[0099] In some aspects, the purification mixture has a temperature from about 0°C to about 12°C (e.g., from about 0°C to about 11°C, about 0°C to about 10°C, about 0°C to about 9°C, from about 0°C to about 8°C, from about 0°C to about 7°C, from about 0°C toabout 6°C, from about 0°C to about 5°C, from about 0°C to about 4°C, from about 0°C to about 3 °C, from about 0°C to about 2°C, from about 0°C to about 1°C, from about 1°C to about 12°C, from about 2°C to about 12°C, from about 3°C to about 12°C, from about 4°C to about 12°C, from about 5°C to about 12°C, from about 6°C to about 12°C, from about 7°C to about 12°C, from about 8°C to about 12°C, from about 9°C to about 12°C, from about 10°C to about 12°C, or from about 11 °C to about 12°C) during the calcium precipitation. In some aspects, the calcium precipitation is conducted at about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, or 12°C. In some aspects, the calcium precipitation is conducted at about 12°C. In some aspects, the calcium precipitation is conducted at about 10°C.[0100| In some aspects, the purification mixture has a temperature from about 10°C to the ambient temperature, e.g., room temperature (e.g., 25°C) during calcium precipitation. In some aspects, the calcium precipitation is conducted from about 10°C to about 24°C, about 10°C to about 23 °C, about 10°C to about 22°C, about 10°C to about 21 °C, about 10°C to about 20°C, about 10°C to about 19°C, about 10°C to about 18°C, about 10°C to about 17°C, about 10°C to about 16°C, about 10°C to about 15°C, about 10°C to about 14°C, about 10°C to about 13°C, about 10°C to about 12°C, about 10°C to about 11°C, about 11°C to about 25°C, about 12°C to about 25°C, about 13°C to about 25°C, about 14°C to about 25°C, about 15°C to about 25°C, about 16°C to about 25°C, about 17°C to about 25°C, about 18°C to about 25°C, about 19°C to about 25°C, about 20°C to about 25°C, about 21°C to about 25°C, about 22°C to about 25°C, about 23°C to about 25°C, or about 24°C to about 25°C. In some aspects, the calcium precipitation is conducted about 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, 20°C, 21°C, 22°C, 23°C, 24°C, or 25°C. In some aspects, the calcium precipitation is conducted at ambient temperature. In some aspects the calcium precipitation is conducted from about 0.5 hours to about 20 hours (e.g., about .5 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 13 hours, about 14 hours, about 15 hours, about 16 hours, about 17 hours, about 18 hours, about 19 hours, or about 20 hours)[0101 [ In some aspects, producing the Kuma polypeptide further comprises one or more processing steps. In some aspects, the one or more processing steps comprise centrifugation, filtration, ultrafiltration / diafiltration, dialyzing, formulation, sterilefiltration, spray drying, packing, storage, or any combination thereof. In some aspects, the one of more processing steps comprises spray drying.

[0012] In some aspects, the Kuma polypeptide is in a formulation buffer during one or more processing steps. The formulation buffer comprises one or more buffering agents. The buffering agent includes, but is not limited to, amino acids, phosphate salt, acetate salt, citrate salt, and Tris. In some aspects, the buffering agent is selected from the group consisting of glycine, glycylglycine, taurine, potassium phosphate, sodium phosphate, potassium acetate, sodium acetate, sodium citrate, Tris buffer. In some aspects, the formulation buffer comprises one or more salt, such as NaCl. In some aspects the formulation buffer comprises one or more excipients. In some aspects, the excipients is a surfactant, such as polysorbate 80. In some aspects, the formulation buffer comprises one or more of the following: Glycine, Glycylglycine, Taurin, Tris buffer, NaCl, polysorbate 80, potassium phosphate, sodium phosphate, sodium acetate, sodium phosphate, and sodium citrate. As used herein, a buffering salt is referred to as a compound of a cation and an anion, which includes all the variants of such salt. For example, in the term sodium phosphate, sodium is the cation and phosphate is the anion. The term includes all the varienty of salts of sodium (Na+) and phosphate (PC>43'), including, but not limited to, monosodium phosphate, disodium phosphate, trisodium phosphate, and the variants thereof.[0103| In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4, about 0.5 to about 5 mM KH2PO4 (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM KH2PO4), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, and about 70 mM NaCl.

[0104] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM Glycine (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM Glycine and about 70 mM NaCl.

[0105] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 5 mM Taurin (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM Taurin), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 27 mM sodium acetate, about 3 mM Taurin, and about 70 mM NaCl.

[0106] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 5 mM Glycylglycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 27 mM sodium acetate, about 3 mM Glycylglycine, and about 70 mM NaCl.[0107 [ In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 5 mM sodium citrate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM sodium citrate), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 27 mM sodium acetate, about 3 mM sodium citrate, and about 70 mM NaCl.

[0108] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM Tris (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Tris), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 13 mM Tris and about 70 mM NaCl.|0109[ In some aspects, the formulation buffer comprises about 10 mM to about 30 mM K2HPO4 (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM K2HPO4), and about 10 mM to about 30 mM Glycine (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM Glycine). In some aspects, the formulation buffer comprises about 20 mM K2HPO4 and about 20 mM Glycine.

[0110] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM K2HPO4 (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM K2HPO4), about 10 mM to about 30 mM Glycine (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 20 mM K2HPO4, about 20 mM Glycine, and about 70 mM NaCl.

[0111] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 0.5 to about 5 mM KH2PO4 (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM KH2PO4), about 20 mM to about 30 mM Glycine (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises 23.37 mM K2HPO4, about 1.63 mM KH2PO4, about 25 mM Glycine, and about 70 mM NaCl.

[0112] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 0.5 to about 5 mM KH2PO4 (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM KH2PO4 about 20 mM to about 40 mM Glycine (e.g., about 20 mM,about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30, mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 28.04 mM K2HPO4, about 1.96 mM KH2PO4, about 30 mM Glycine, and about 70 mM NaCl.

[0113] In some aspects, the formulation buffer comprises about 20 mM to about 40 mM Glycine (e.g., about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30, mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 30 mM Glycine and about 70 mM NaCl.

[0114] In some aspects, the formulation buffer comprises about 40 mM to about 60 mM Glycine (e.g., about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50, mM, about 52 mM, about 54 mM, about 56 mM, about 58 mM, or about 60 mM Glycine) and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 50 mM Glycine and about 70 mM NaCl.

[0115] In some aspects, the formulation buffer comprises about 40 mM to about 50 mM sodium acetate (e.g., about 40 mM about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM sodium acetate), about 1 mM to about 10 mM Glycylglycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 45 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl.

[0116] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 10 mM Glycylglycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl).In some aspects, the formulation buffer comprises about 25 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl.

[0117] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM sodium acetate (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM sodium acetate), about 1 mM to about 20 mM Glycylglycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl.

[0118] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 1 mM to about 20 mM Glycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM K2HPO4, about lOmM Glycine, and about 70 mM NaCl.[0119 [ In some aspects, the formulation buffer comprises about 20 mM to about 30 mM Na2HPC>4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM Na2HPC>4), about 1 mM to about 10 mM Glycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM Na2HPC>4, about 5mM Glycine, and about 70 mM NaCl.

[0120] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM sodium acetate (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM sodium acetate), about 1 mM to about 20 mM Glycylglycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, orabout 80 mM NaCl). In some aspects, the formulation buffer comprises about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl.

[0121] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM Tris (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Tris), about 0.01% to about 0.10% polysorbate 80 (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.10% polysorbate 80), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 13 mM Tris, about 0.05% polysorbate 80, and about 70 mM NaCl.

[0122] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM Tris (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Tris) and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 13 mM Tris and about 70 mM NaCl.

[0123] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM K2HPO4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mMK2HPO4), about 10 mM to about 20 mM Na2HPC>4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Na2HPC>4), about 1 mM to about 20 mMGlycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.

[0124] In some aspects, the Kuma polypeptide is in a formulation buffer selected from the group consisting of about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, and about 70 mM NaCl; about 25 mM Glycine and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Taurin, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Glycylglycine, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mMsodium citrate, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; about 20 mM K2HPO4 and about 20 mM Glycine; about 20 mM K2HPO4, about 20 mM Glycine, and about 70 mM NaCl; about 23.37 mM BGHPC , about 1.63 mM KTbPC , about 25 mM Glycine, and about 70 mM NaCl; about 28.04 mM K2HPO4, about 1.96 mM KH2PO4, about 30 mM Glycine, and about 70 mM NaCl; about 30 mM Glycine and about 70 mM NaCl; about 50 mM Glycine and about 70 mM NaCl; about 45 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; 25 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 25 mM K2HPO4, about lOmM Glycine, and about 70 mM NaCl; about 25 mM Na2HPC>4, about 5mM Glycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 13 mM Tris, about 0.05% polysorbate 80, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; and about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.

[0125] In some aspects, the formulation buffer has a pH from about 7.2 to about 10.2. In some aspects, the formulation buffer has a pH of about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, or about 10.2. In some aspects, the formulation buffer has a pH of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10.0. In particular aspects, the formulation buffer has a pH of about 8.0. In particular aspects, the formulation buffer has a pH of about 8.5. In particular aspects, the formulation buffer has a pH of about 9.0.(0126] In some aspects, the formulation buffer further comprises from about 20% to about 50% trehalose (e.g., from about 25% to about 45%, from about 30% to about 40%, or about 35%). In some aspects, the formulation buffer further comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose. In some aspects, the formulation buffer further comprises about 25% trehalose. In some aspects, the formulation buffer further comprises about 47% trehalose.|0127| In some aspects, the processing step comprising spray drying comprises a spraydrying solution. In some aspects, the spray-drying solution comprises silica. In some aspects, the spray-drying solution does not comprise silica.

[0128] In some aspects, the concentration of the Kuma polypeptide is from about 1 g / L to about 150 g / L (e.g., from about 5 g / L to about 150 g / L, about 10 g / L to about 150 g / L, about 20 g / L to about 150 g / L, about 30 g / L to about 150 g / L, about 40 g / L to about 150 g / L, about 50 g / L to about 150 g / L, about 60 g / L to about 150 g / L, about 70 g / L to about 150 g / L, about 80 g / L to about 150 g / L, about 90 g / L to about 150 g / L, about 100 g / L to about 150 g / L, about 110 g / L to about 150 g / L, about 120 g / L to about 150 g / L, about 130 g / L to about 150 g / L, about 140 g / L to about 150 g / L, about 1 g / L to about 140 g / L, about 1 g / L to about 130 g / L, about 1 g / L to about 120 g / L, about 1 g / L to about 110 g / L, about 1 g / L to about 100 g / L, about 1 g / L to about 90 g / L, about 1 g / L to about 80 g / L, about 1 g / L to about 70 g / L, about 1 g / L to about 60 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 40 g / L, about 1 g / L to about 30 g / L, about 1 g / L to about 20 g / L, about 1 g / L to about 10 g / L, or about 1 g / L to about 5 g / L.) In some aspects, the concentration of the Kuma polypeptide is about 1 g / L, about 5 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, or about 150 g / L.101291 The present disclosure provides methods of producing a Kuma polypeptide, and compositions comprising a Kuma polypeptide, wherein the Kuma polypeptide comprises a polypeptide or variant thereof having at least 75% (e.g., having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9. In some aspects, the Kuma polypeptide comprises a polypeptide or variant thereof having at least 75% (e.g., having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the Kuma polypeptide comprises Kuma062 or a variant thereof. In a particular aspect, the Kuma polypeptide is a polypeptide or variant thereof comprising or consisting of SEQ ID NO: 1.Table 1: Kuma Sequences

[0130] A Kuma polypeptide may have two versions, an unprocessed version and a processed version. The unprocessed version, sometimes referred to as full length Kuma, comprises a pre-protein domain, which includes the N-terminal portion of the full length Kuma. The processed version, sometimes referred to as mature Kuma, includes the portion of full length Kuma after the removal of the pre-protein domain from the full length Kuma. The unprocessed and processed versions of a Kuma may have one or more variants. For example, as shown in Table 1 above, SEQ ID NO: 14 represents a variant of mature KumaOl 1. SEQ ID NO: 1 is the unprocessed version of Kuma062; SEQ ID NO: 10 is the pre-protein domain; SEQ ID NO: 11 is the processed version of Kuma062; SEQ ID NO: 12 is the processed version of Kuma062 minus the C-terminal Proline residue. SEQ ID NO: 2 is the unprocessed version of KumaOl 1; SEQ ID NO: 13 is the processed version of Kuma062; SEQ ID NO: 14 is the processed version of KumaOl 1 minus the C- terminal Proline residue. SEQ ID NO: 3 is the unprocessed version of Kuma021; SEQ ID NO: 15 is the processed version of Kuma021. SEQ ID NO: 4 is the unprocessed version of Kuma031; SEQ ID NO: 16 is the processed version of Kuma031. SEQ ID NO: 5 is theunprocessed version of Kuma041; SEQ ID NO: 17 is the processed version of Kuma041. SEQ ID NO: 6 is the unprocessed version of Kuma051; SEQ ID NO: 18 is the processed version of Kuma051. SEQ ID NO: 7 is the unprocessed version of Kuma061; SEQ ID NO: 19 is the processed version of Kuma061. SEQ ID NO: 8 is the unprocessed version of Kuma071; SEQ ID NO: 20 is the processed version of Kuma071. SEQ ID NO: 9 is the unprocessed version of Kuma072; SEQ ID NO: 21 is the processed version of Kuma072.

[0131] In some aspects, a method disclosed herein comprises suspending one or more host cells comprising the polypeptide in a cell suspension; heat treating the cell suspension; and purifying the Kuma polypeptide by calcium precipitation; and wherein the Kuma polypeptide comprises the amino acid sequence set forth in SEQ ID NO 1. In some aspects, the cell suspension has a pH of about 9.2. In some aspects, cell suspension comprises Arg-buffer, wherein the Arg-buffer comprises about 3.4 mmol / L CaCh. In some aspects, the heat treatment further comprises a cooling step.

[0132] In some aspects, a method disclosed herein comprises comprises suspending one or more host cells comprising the polypeptide in an Arg-buffer, thereby producing a cell suspension, wherein the Arg-buffer comprises about 0.58 g / L sodium chloride, about 4.2 g / L L- Arginine salt, and about 3.4 mmol / L calcium chloride; heating treating the cell suspension to about 65°C for about 1 minute; and cooling the cell suspension after heating treatment to about 10°C.

[0133] In some aspects, the Kuma polypeptide is a Kuma062 variant, which comprises an amino acid sequence at least 75% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) identical to the amino acid sequence of SEQ ID NO: 1 wherein residue 466 is Ser, residue 266 is Glu, and residue 270 is Asp; and the polypeptide comprises an amino acid at one or more residues selected from the group consisting of 220D / N / Q / H, 26 IE, 267S / T / A, 268L / T, 269A / T / V, 318A, 319M, 353A / E / Q / R / Y, 357N / S / Q / T, 367F / Q, 398Q, 401 S / Q, 405S, 423K, 448E / N / Q, 460R, and 462A / L / M / Q / R / T / V. In some aspects, the Kuma062 variant comprises an amino acid at two or more residues selected from the group consisting of 220D / N / Q / H, 261E, 267S / T / A, 268L / T, 269A / T / V, 318A, 319M, 353A / E / Q / R / Y, 357N / S / Q / T, 367F / Q, 398Q, 401 S / Q, 405S, 423K, 448E / N / Q, 460R, and 462A / L / M / Q / R / T / V. In some aspects, the Kuma062 variant comprises amino acids 357S and 462T, optionally, the Kuma062 variant further comprises 398Q and 448Q. In some aspects, the Kuma062 variantcomprises amino acid 261E, 268T, 353Q, 357S, 398Q, 448Q, and 462T, optionally, the polypeptide further comprises 319M. In some aspects, the Kuma062 variant comprises amino acid 261E, 268T, 269V, 353Q, 357S, 398Q, and 448Q. In some aspects, the Kuma062 variant comprises amino acid 318A, 367F, 398Q, 448Q, and 462T, optionally, the polypeptide further comprises 319M. In some aspects, the Kuma062 variant further comprises an amino acid at one or more amino acid positions selected from the group consisting of 104H; 170R / A / S; 171R / A / S; 172R / S, 173S, and / or 455V.

[0134] In some aspects, a variant of a Kuma062 comprises an amino acid sequence as set forth in SEQ ID No: 22.Compositions of the Disclosure

[0135] Some aspects of the present disclosure provide compositions comprising a Kuma polypeptide.

[0136] In some aspects, the composition comprises a Kuma polypeptide and a formulation buffer. The formulation buffer can be any formulation buffer described herein.

[0137] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 0.5 to about 5 mM KH2PO4 (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM KH2PO4), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, and about 70 mM NaCl.

[0138] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM Glycine (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM Glycine and about 70 mM NaCl.|0139| In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 5 mM Taurin (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM Taurin), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 27 mM sodium acetate, about 3 mM Taurin, and about 70 mM NaCl.

[0140] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 5 mM Glycylglycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 27 mM sodium acetate, about 3 mM Glycylglycine, and about 70 mM NaCl.

[0141] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 5 mM sodium citrate (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, or about 5 mM sodium citrate), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 27 mM sodium acetate, about 3 mM sodium citrate, and about 70 mM NaCl.

[0142] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM Tris (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Tris), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 13 mM Tris and about 70 mM NaCl.

[0143] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM K2HPO4 (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM K2HPO4), and about 10 mM to about 30 mM Glycine (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24mM, about 26 mM, about 28 mM, or about 30 mM Glycine). In some aspects, the formulation buffer comprises about 20 mM K2HPO4 and about 20 mM Glycine.

[0144] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM K2HPO4 (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM K2HPO4), about 10 mM to about 30 mM Glycine (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20, mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 20 mM K2HPO4, about 20 mM Glycine, and about 70 mM NaCl.

[0145] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 0.5 to about 5 mM KH2PO4 (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM KH2PO4), about 20 mM to about 30 mM Glycine (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises 23.37 mM K2HPO4, about 1.63 mM KH2PO4, about 25 mM Glycine, and about 70 mM NaCl.|0146[ In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 0.5 to about 5 mM KH2PO4 (e.g., about 0.5 mM, about 1 mM, about 1.5 mM, about 2 mM, about 2.5 mM, about 3 mM, about 3.5 mM, about 4 mM, about 4.5 mM, or about 5 mM KH2PO4 about 20 mM to about 40 mM Glycine (e.g., about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30, mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 28.04 mM K2HPO4, about 1.96 mM KH2PO4, about 30 mM Glycine, and about 70 mM NaCl.101471 In some aspects, the formulation buffer comprises about 20 mM to about 40 mMGlycine (e.g., about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, about 30, mM, about 32 mM, about 34 mM, about 36 mM, about 38 mM, or about 40 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 30 mM Glycine and about 70 mM NaCl.

[0148] In some aspects, the formulation buffer comprises about 40 mM to about 60 mM Glycine (e.g., about 40 mM, about 42 mM, about 44 mM, about 46 mM, about 48 mM, about 50, mM, about 52 mM, about 54 mM, about 56 mM, about 58 mM, or about 60 mM Glycine) and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 50 mM Glycine and about 70 mM NaCl.

[0149] In some aspects, the formulation buffer comprises about 40 mM to about 50 mM sodium acetate (e.g., about 40 mM about 41 mM, about 42 mM, about 43 mM, about 44 mM, about 45 mM, about 46 mM, about 47 mM, about 48 mM, about 49 mM, or about 50 mM sodium acetate), about 1 mM to about 10 mM Glycylglycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 45 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl.(0150] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM sodium acetate (e.g., about 20 mM about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM sodium acetate), about 1 mM to about 10 mM Glycylglycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl.

[0151] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM sodium acetate (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about30 mM sodium acetate), about 1 mM to about 20 mM Glycylglycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl.

[0152] In some aspects, the formulation buffer comprises about 20 mM to about 30 mM K2HPO4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM K2HPO4), about 1 mM to about 20 mM Glycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM K2HPO4, about lOmM Glycine, and about 70 mM NaCl.10153] In some aspects, the formulation buffer comprises about 20 mM to about 30 mMNa2HPC>4 (e.g., about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25, mM, about 26 mM, about 27 mM, about 28 mM, about 29 mM, or about 30 mM Na2HPC>4), about 1 mM to about 10 mM Glycine (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, or about 10 mM Glycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 25 mM Na2HPC>4, about 5mM Glycine, and about 70 mM NaCl.

[0154] In some aspects, the formulation buffer comprises about 10 mM to about 30 mM sodium acetate (e.g., about 10 mM, about 12 mM, about 14 mM, about 16 mM, about 18 mM, about 20 mM, about 22 mM, about 24 mM, about 26 mM, about 28 mM, or about 30 mM sodium acetate), about 1 mM to about 20 mM Glycylglycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM Glycylglycine), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl.

[0155] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM Tris (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Tris),about 0.01% to about 0.10% polysorbate 80 (e.g., about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, or about 0.10% polysorbate 80), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 13 mM Tris, about 0.05% polysorbate 80, and about 70 mM NaCl.

[0156] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM Tris (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Tris) and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 13 mM Tris and about 70 mM NaCl.

[0157] In some aspects, the formulation buffer comprises about 10 mM to about 20 mM K2HPO4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM K2HPO4), about 10 mM to about 20 mM Na2HPC>4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Na2HPC>4), about 1 mM to about 20 mM Glycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.

[0158] In some aspects, the formulation buffer is selected from the group consisting of about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, and about 70 mM NaCl; about 25 mM Glycine and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Taurin, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM Glycylglycine, and about 70 mM NaCl; about 27 mM sodium acetate, about 3 mM sodium citrate, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; about 20 mM K2HPO4 and about 20 mM Glycine; about 20 mM K2HPO4, about 20 mM Glycine, and about 70 mM NaCl; about 23.37 mM K2HPO4, about 1.63 mM KH2PO4, about 25 mM Glycine, and about 70 mM NaCl; about 28.04 mM K2HPO4, about 1.96 mM KH2PO4, about 30 mM Glycine, and about 70 mM NaCl; about 30 mM Glycine and about 70 mM NaCl; about50 mM Glycine and about 70 mM NaCl; about 45 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; 25 mM sodium acetate, about 5 mM Glycylglycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 25 mM K2HPO4, about lOmM Glycine, and about 70 mM NaCl; about 25 mM Na2HPC>4, about 5mM Glycine, and about 70 mM NaCl; about 20 mM sodium acetate, about 10 mM Glycylglycine, and about 70 mM NaCl; about 13 mM Tris, about 0.05% polysorbate 80, and about 70 mM NaCl; about 13 mM Tris and about 70 mM NaCl; and about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.|0159| In some aspects, the formulation buffer has a pH from about 7.2 to about 10.2. In some aspects, the formulation buffer has a pH of about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, or about 10.2. In some aspects, the formulation buffer has a pH of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10.0. In particular aspects, the formulation buffer has a pH of about 8.0. In particular aspects, the formulation buffer has a pH of about 8.5. In particular aspects, the formulation buffer has a pH of about 9.0.

[0160] In some aspects, the composition comprises a Kuma polypeptide and a formulation buffer comprising about 10 mM to about 20 mM K2HPO4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM K2HPO4), about 10 mM to about 20 mM Na2HPC>4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Na2HPC>4), about 1 mM to about 20 mM Glycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl. In some aspects, the composition comprises a Kuma polypeptide and a formulation buffercomprising about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl.

[0161] In some aspects, the formulation buffer further comprises from about 20% to about 50% trehalose (e.g., from about 25% to about 45%, from about 30% to about 40%, or about 35%). In some aspects, the formulation buffer further comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose. In some aspects, the formulation buffer further comprises about 25% trehalose. In some aspects, the formulation buffer further comprises about 47% trehalose. In some aspects, the formulation buffer does not comprise trehalose.101621 In some aspects, the composition further comprises silica. In some aspects, the composition does not comprise silica.

[0163] In some aspects, the concentration of the Kuma polypeptide is from about 1 g / L to about 150 g / L (e.g., from about 5 g / L to about 150 g / L, about 10 g / L to about 150 g / L, about 20 g / L to about 150 g / L, about 30 g / L to about 150 g / L, about 40 g / L to about 150 g / L, about 50 g / L to about 150 g / L, about 60 g / L to about 150 g / L, about 70 g / L to about 150 g / L, about 80 g / L to about 150 g / L, about 90 g / L to about 150 g / L, about 100 g / L to about 150 g / L, about 110 g / L to about 150 g / L, about 120 g / L to about 150 g / L, about 130 g / L to about 150 g / L, about 140 g / L to about 150 g / L, about 1 g / L to about 140 g / L, about 1 g / L to about 130 g / L, about 1 g / L to about 120 g / L, about 1 g / L to about 110 g / L, about 1 g / L to about 100 g / L, about 1 g / L to about 90 g / L, about 1 g / L to about 80 g / L, about 1 g / L to about 70 g / L, about 1 g / L to about 60 g / L, about 1 g / L to about 50 g / L, about 1 g / L to about 40 g / L, about 1 g / L to about 30 g / L, about 1 g / L to about 20 g / L, about 1 g / L to about 10 g / L, or about 1 g / L to about 5 g / L.) In some aspects, the concentration of the Kuma polypeptide is about 1 g / L, about 5 g / L, about 10 g / L, about 20 g / L, about 30 g / L, about 40 g / L, about 50 g / L, about 60 g / L, about 70 g / L, about 80 g / L, about 90 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, or about 150 g / L. In some aspects, the concentration is about 100 g / L. In some aspects, the concentration is about 2 g / L.

[0164] In some aspects, the Kuma polypeptide comprises a polypeptide or variant thereof having at least 75% e.g., having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9. In some aspects, the Kumapolypeptide comprises a polypeptide or variant thereof having at least 75% (e.g., having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the Kuma polypeptide comprises Kuma062 or a variant thereof. In a particular aspect, the Kuma polypeptide is a polypeptide or variant thereof comprising or consisting of SEQ ID NO: 1.

[0165] In some aspects, the present disclosure provides a dry powder subtance comprising a Kuma polypeptide. In some aspects, the dry powder substance is prepared through any method described herein. For example, the dry powder substance may be prepared via heat treatment, flocculation, and calcium prepcipitation described herein. In some aspects, the dry powder substance is prepared by spray drying a Kuma polypeptide in any formulation buffer described herein. In some aspects, the dry powder substance comprises silica. In some aspects, the dry powder substance does not comprise silica. In some aspects, the dry powder substance comprises from about 0.5% to about 2.4% (w / w) silica. In some aspects, the dry powder substance comprises about 1.36% (w / w) silica. In some aspects, the silica in the dry powder substance is hydrophilic fumed silica (Cab-O- Sil).Processes of the Disclosure

[0166] Some aspects of the present disclosure provide processes for producing a dry powder substance comprising a Kuma polypeptide.10167] In some aspects, the process for producing a dry powder drug substance comprising a Kuma polypeptide comprises suspending one or more host cells comprising the Kuma polypeptide in a cell suspension buffer; heat-treating the cell suspension; flocculating the cell suspension; purifying the Kuma polypeptide by calcium precipitation; concentrating the Kuma polypeptide in a solution; changing the buffer in the concentrated solution to a formulation buffer, thereby producing a spray-drying solution comprising the Kuma polypeptide in the formuation buffer; and spray-drying the spray-drying solution. Each step of the above process can be performed using any method or process described herein.|0168| In some aspects, the spray-drying solution comprises silica. In some aspects, the spray-drying solution does not comprise silica.101691 In some aspects, the cell suspension comprises Ca2+. In some aspects, the cell suspension buffer comprises from about 1 mmol / L CaCh to about 10 mmol / L CaCh (e.g., from about 1 mmol / L to about 9 mmol / L, from about 2 mmol / L to about 9 mmol / L, from about 3 mmol / L to about 9 mmol / L, from about 4 mmol / L to about 9 mmol / L, from about 5 mmol / L to about 9 mmol / L, from about 6 mmol / L to about 9 mmol / L, from about 7 mmol / L to about 9 mmol / L, from about 8 mmol / L to about 9 mmol / L, from about 1 mmol / L to about 8 mmol / L, from about 1 mmol / L to about 7 mmol / L, from about 1 mmol / L to about 6 mmol / L, from about 1 mmol / L to about 5 mmol / L, from about 1 mmol / L to about 4 mmol / L, from about 1 mmol / L to about 3 mmol / L, or from about 1 mmol / L to about 2 mmol / L). In some aspects, the cell suspension buffer comprises about 1 mmol / L CaCh, about 1.5 mmol / L CaCh, about 2 mmol / L CaCh, about 2.5 mmol / L CaCh, about 3 mmol / L CaCh, about 3.5 mmol / L CaCh, about 4 mmol / L CaCh, about 4.5 mmol / L CaCh, about 5 mmol / L CaCh, about 5.5 mmol / L CaCh, about 6 mmol / L CaCh, about 6.5 mmol / L CaCh, about 7 mmol / L CaCh, about 7.5 mmol / L CaCh, about 8 mmol / L CaCh, about 8.5 mmol / L CaCh, about 9 mmol / L CaCh, about 9.5 mmol / L CaCh, or about 10 mmol / L CaCh. In some aspects, the cell suspension buffer comprises about 3.4 mmol / L CaCh.

[0170] In some aspects, the heat treatment comprises heating the cell suspension to about 40°C to about 90°C (e.g., to about 40°C to about 80°C, to about 40°C to about 70°C, to about 40°C to about 60°C, to about 40°C to about 50°C, to about 50°C to about 90°C, to about 50°C to about 80°C, to about 50°C to about 70°C, or to about 50°C to about 60°C) for about 5 seconds to about 15 minutes (e.g., from about 5 seconds to about 14 minutes, from about 5 seconds to about 13 minutes, from about 5 seconds to about 12 minutes, from about 5 seconds to about 11 minutes, from about 5 seconds to about 10 minutes, from about 5 seconds to about 9 minutes, from about 5 seconds to about 8 minutes, from about 5 seconds to about 7 minutes, from about 5 seconds to about 6 minutes, from about 5 seconds to about 5 minutes, from about 5 seconds to about 4.5 minutes, from about 5 seconds to about 4 minutes, from about 5 seconds to about 3.5 minutes, from about 5 seconds to about 3 minutes, from about 5 seconds to about 2.5 minutes, from about 5 seconds to about 2 minutes, from about 5 seconds to about 1.5 minutes, from about 5 seconds to about 1 minute, from about 5 seconds to about 30 seconds, from about 5 seconds to about 10 seconds, from about 10 seconds to about 15 minutes, from about 30 seconds to about 15 minutes, from about 1 minute to about 15 minutes, from about 1.5minutes to about 15 minutes, from about 2 minutes to about 15 minutes, from about 2.5 minutes to about 15 minutes, from about 3 minutes to about 15 minutes, from about 3.5 minutes to about 15 minutes, from about 4 minutes to about 15 minutes, from about 4.5 minutes to about 15 minutes, from about 5 minutes to about 15 minutes, from about 6 minutes to about 15 minutes, from about 7 minutes to about 15 minutes, from about 8 minutes to about 15 minutes, from about 9 minutes to about 15 minutes, from about 10 minutes to about 15 minutes, from about 11 minutes to about 15 minutes, from about 12 minutes to about 15 minutes, from about 13 minutes to about 15 minutes, or from about 14 minutes to about 15 minutes). In some aspects, the heat treatment comprises heating the cell suspension to about 50°C to about 80°C for about 5 seconds to about 3 minutes. In some aspects, the heat treatment comprises heating the cell suspension to about 60°C to about 70°C for about 1 minute to about 2 minutes. In some aspects, the heat treatment comprises heating the cell suspension to about 60°C to about 70°C for about 1 minute. In particular aspects, the heat treatment comprises heating the cell suspension to about 65°C for about 1 minute.[01711 In some aspects, the heat treatment further comprises a cooling step. In some aspects, the cooling step comprises cooling the cell suspension to about 0°C to about 15°C (e.g., from about 0°C to about 14°C, from about 0°C to about 13°C, from about 0°C to about 12°C, from about 0°C to about 11°C, from about 0°C to about 10°C, from about 0°C to about 9°C, from about 0°C to about 8°C, from about 0°C to about 7°C, from about0°C to about 6°C, from about 0°C to about 5°C, from about 0°C to about 4°C, from about0°C to about 3 °C, from about 0°C to about 2°C, from about 0°C to about 1°C, from about1°C to about 15°C, from about 2°C to about 15°C, from about 3°C to about 15°C, from about 4°C to about 15°C, from about 5°C to about 15°C, from about 6°C to about 15°C, from about 7°C to about 15°C, from about 8°C to about 15°C, from about 9°C to about 15°C, from about 10°C to about 15°C, from about 11°C to about 15°C, from about 12°C to about 15°C, from about 13°C to about 15°C, or from about 14°C to about 15°C). In some aspects, the cooling step comprises actively cooling the cell suspension or purification mixture to about 0°C, 1°C, 2°C, 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, 15°C, 16°C, 17°C, 18°C, 19°C, or 20°C. In some aspects, the cooling step comprises actively cooling the cell suspension to about 0°C. In some aspects, the cooling step comprises actively cooling the cell suspension to about 5°C. In some aspects, the cooling step comprises actively cooling the cell suspension to about 10°C. Insome aspects, the cooling step comprises actively cooling the cell suspension to about 15°C. In some aspects, the cooling step comprises actively cooling the cell suspension to about 20°C.

[0172] In some aspects, the cell suspension has a pH from about 7.2 to about 10.2. In some aspects, the cell suspension has a pH of about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, about 10.0, about 10.1, or about 10.2 during the heat treatment. In some aspects, the cell suspension has a pH of about 8.5, about 8.6, about 8.7, about 8.8, about 8.9, about 9.0, about 9.1, about 9.2, about 9.3, about 9.4, about 9.5, about 9.6, about 9.7, about 9.8, about 9.9, or about 10.0. In particular aspects, the cell suspension has a pH of about 9.4.

[0173] In some aspects, the formulation buffer is a phosphate-glycine buffer. In some aspects, the phosphate-glycine buffer comprises about 10 mM to about 20 mM K2HPO4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM K2HPO4), about 10 mM to about 20 mM Na2HPC>4 (e.g., about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, or about 20 mM Na2HPC>4), about 1 mM to about 20 mM Glycine (e.g., about 1 mM, about 5 mM, about 10 mM, about 15 mM, or about 20 mM), and about 60 to about 80 mM NaCl (e.g., about 60 mM, about 65 mM, about 70 mM, about 75 mM, or about 80 mM NaCl). In some aspects, the formulation buffer comprises about 12.5 mM K2HPO4, about 12.5 mM Na2HPC>4, about 10 mM Glycine, and about 70 mM NaCl. In some aspects, the phosphate-glycine buffer comprises 12.5 mM K2HPO4, 12.5 mM Na2HPC>4, 10 mM Glycine, and 70 mM NaCl.

[0174] In some aspects, formulation buffer further comprises from about 20% to about 50% trehalose (e.g., from about 25% to about 45%, from about 30% to about 40%, or about 35%). In some aspects, the formulation buffer further comprises about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% trehalose. In some aspects, the formulation buffer further comprises about 25% trehalose. In some aspects, the formulation buffer further comprises about 47% trehalose.10.175] In some aspects, the formulation buffer comprises silica. In some aspects, the formulation buffer does not comprise silica.101.76| In some aspects, the Kuma polypeptide comprises a polypeptide or variant thereof having at least 75% (e.g., having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity to an amino acid sequence set forth in any one of SEQ ID NOs: 1-9. In some aspects, the Kuma polypeptide comprises a polypeptide or variant thereof having at least 75% (e.g., having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%) sequence identity to an amino acid sequence set forth in SEQ ID NO: 1. In some aspects, the Kuma polypeptide comprises Kuma062 or a variant thereof. In a particular aspect, the Kuma polypeptide is a polypeptide or variant thereof comprising or consisting of SEQ ID NO: 1.EXAMPLES

[0177] The example presented below is provided for the purpose of illustration only, and the aspects described herein should in no way be construed as being limited to this example. Rather, the aspects should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.Example 1: Production of Kuma062

[0178] Kuma062, the polypeptide with the amino acid sequence set forth in SEQ ID NO: 1, was produced using the processes shown in FIG. 1. A vial of E. coli transfected to stably express Kuma062 was thawed and cultured in shake flasks at 32°C in suitable culture media. Then, the culture was transferred to a main fermentor set at 32°C. The temperature at the main fermentor was adjusted to 25°C. The cell culture medium used in the main fermentation can optionally include about 3.4 mmol / L calcium chloride.

[0179] After fermentation, the broth was washed and diluted in Arg-buffer, containing 0.58 g / L sodium chloride, 4.2 g / L L- Arginine salt, and 3.4 mmol / L calcium chloride, with a pH value of 8.2 ± 0.2, to a wet cell weight (WCW) of 10%. The pH was adjusted to about 9.2 using sodium hydroxide and kept between 5-10°C, and then centrifuged. The cell pellet was resuspended in Arg-buffer, containing 3.4 mmol / L calcium chloride, to a WCW of 10%. The cell suspension was then heat treated by heating to between about60°C to about 70°C (e.g., to about 65°C) for about 1 minute and then quickly cooled back to about 10°C. The heat treatment process required two heat exchangers, for heating and for cooling, so that it was an in-process heat treatment.

[0180] Flocculation, a process of aggregating dispersed microbial cells, was performed by addition of poly-s-lysine dissolved in Arg-buffer (final concentration of poly-s-lysine: 2 g / L) at a temperature of <10°C and a pH about 7.5. Impurities formed as solid precipitate after about 6 hours or more. The mixture was centrifuged to separate solid impurities at a temperature of <10°C.10181] The mixture was further precipitated by addition of a phosphate stock solution (350 mmol / L phosphate stock solution) with a pH between 7.2-9.2. The final concentration of phosphate was 35 mmol / L. After mixing for about 15 minutes, calcium acetate was sequentially added up to 10 times and continuously agitated. The final concentration of calcium acetate was 70 mmol / L. Upon each addition, the pH of the mixture dropped, and the pH of the mixture was adjusted continuously to maintain a pH between 7.2-9.2 by addition of sodium hydroxide. The mixture was stirred for more than 12 hours at a temperature of <10°C. Then, the mixture was centrifuged and filtered at a temperature <12°C. Ultrafiltration / Diafiltration was performed at ambient temperature (18-25 °C) using ultrafiltration membranes with a molecular weight cutoff (MWCO) of 50 kDa. The filtrate obtained was concentrated to a desired concentration of 60 g / L, followed by diafiltration, before a final desired concentration of 110 g / L was reached.

[0182] At ambient temperature, the filtered substance was stabilized and formulated by addition of trehalose and polysorbate 80. The substance was then sterile filtered and spray dried using a two-nozzle spray dryer / cyclone at an inlet temperature of 135-145 °C. The spray dried powder was packed under sterile conditions and stored at 2-8 °C.Example 2: Trehalose reduction and spray drying development

[0183] An alternative buffer system and reduction of trehalose content for the Kuma062 spray dried drug formulation was investigated. Screening experiments with different buffer chemicals were performed to find an alternative buffer substance to Tris buffer (13 mM Tris, 70 mM NaCl, pH 8.0). Also, possible ways to reduce the solid content of the formulation to minimize tablet size were investigated. Therefore, the spray drying process and stability of the Kuma062 spray dried drug formulation with no or reduced trehalose concentration was evaluated.101.841 Several buffer systems were evaluated for buffering in the alkaline range (pH>8.0) as an alternative to Tris buffer. The stability studies were performed with high (approx. 100 g / L) and low (approx.. 2 g / L) Kuma062 protein concentration, and a buffer pH study was undertaken without Kuma062 protein.101851 In a first experiment, Kuma062 protein at 100 g / L was dialyzed in the respective buffers (shown in Table 2), and the liquid drug substance stability was evaluated in stability studies. The study samples were stored at +15°C and +30°C up to 28 days. Samples were taken for analysis (size exclusion chromatography (SEC); dynamic light scattering (DLS) analysis; differential scanning fluorimetry (DSF) analysis; enzyme activity assay; protein concentration determination; pH determination; SDS-PAGE and Western blot analysis) at day 0, day 7, day 14 and day 21 or day 28.

[0186] The first study launched to find an alternative buffer candidate, included the evaluation of five potential suitable buffer systems listed in Table 2. Buffers buffering in the alkaline range were tested and also an acidic component combined with alkaline component (e.g. sodium acetate with Taurin) were tested. An additional experiment was started after the observation of pH instability of the samples after dialysis. The pH values of buffer only were determined when stored at +15°C, +30°C or RT while exposed to air for 2 weeks, to examine the buffering capacity.Table 2: Buffer screening 1 (high protein concentration)

[0187] Kuma062 was stored in the respective buffers (Table 2) at +15°C and +30°C up to 28 days. The stability was evaluated. From this study it was concluded that Kuma062 is a very stable protein and all 6 tested buffer systems only showed little differences. No significant difference was observed with these six buffers. Experiments continued with buffers 01 (Phosphate), 02 (Glycine NaOH) and 04 (Acetate + GlyGly) because they had the best performance in DLS, SEC and AEX analysis. From this study, it was concluded that Kuma062 is a very stable protein and all six tested buffer systems only showed little differences at high protein concentration (lOOg / L).

[0188] Consequently, the stability of three buffer systems (phosphate, Glycine NaOH, Acetat + GlyGly) were tested in different excipient concentrations in absence of Kuma062 protein (Table 3). Glycine was added to the phosphate buffer samples as glycine showed beneficial effects on the stability of Kuma062 protein.

[0189] The second study examined the pH stability of the respective buffers with and without air exposure.Table 3: Buffers used in in study 2[0.190] The buffers were incubated at +30°C for 14 days as well as at room temperature with and without exposure to air. The results indicated that among the 4 different phosphate buffers with glycine (01-04), 01 and 03 (23.37 mM K2HPO4; 1.63 mM KH2PO4; 25 mM Glycine; pH 8.5, containing 0 or 70 mM NaCl) were the most stable. Due to additional of salt, which is known to have a positive effect on protein stability, buffer 03 was preferred over buffer 01. Among acetate GLY-GLY buffers (07-09), 09 with the highest GLY-GLY concentration (10 mM GLY-GLY) was the most stable buffer.

[0191] In the third and final study, stability of Kuma062 protein at 2 g / L in prescence of phosphate buffer + glycine, Acetate + GLY-GLY and Tris buffer with and without polysorbate 80 was evaluated (Table 4) using stability indicating methods as in previous studies. Storage at +4°C, +15°C and 40°C for 14 days and 21 days was conducted.

[0192] To simplify buffer preparation for production purposes, the ratio of phosphate to glycine was optimized to obviate the need for an additional pH adjustment step, ensuring that the buffer's inherent pH aligns with the target pH.

[0193] As a control, a pH stability study was conducted with buffers used in the stabilityTable 4: Buffers used in study 3

[0014] The analytical differences between the 5 tested buffer systems were minimal. Acetate + Gly-Gly buffer was excluded due to most significant impacts on aggregate formation, loss of activity, and pH instability. The two phosphate buffers were able to stabilize Kuma062 in the same matter as Tris buffer. This study also revealed that addition of polysorbate 80 did not add stability.

[0195] Experiments were then performed to evaluate the possibility for trehalose reduction without impairing Kuma062 drug substance quality and stability. Kuma062 concentrated liquid material with a concentration of approximately 100 g / L was dialyzed against alternative “Phosphate buffer” to perform spray drying with reduced trehalose content or without trehalose. No dialysis was done with experiments with “Tris buffer” experiments.10196] Both, Potassium Phosphate Glycine buffer and Sodium Phosphate Glycine buffer provided good conditions for stabilizing Kuma062 protein at +4°C, +15°C and 40°C and were chosen as alternative drug substance formulations to Tris buffer. The final drug substance buffer for the formulation was the “Phosphate buffer”: Potassium-Sodium- Phosphate Glycine buffer (12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, 70 mM NaCl pH 8.5.Trehalose Reduction Studies

[0197] Kuma062 was formulated and spray-dried using two novel drug substance formulation options - Phase 2 “Tris buffer”: 13 mM Tris, 70 mM NaCl, pH 8.0 with reduced (25%) trehalose and “Phosphate buffer”: K-Na-Phosphate Glycine buffer (12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, 70 mM NaCl pH 8.5) with reduced (25%) trehalose content.

[0198] Experiments were performed to evaluate the possibility of trehalose reduction without impairing Kuma062 drug substance quality and stability. Kuma062 concentrated liquid material with a concentration of approximately 100 g / L was dialyzed against alternative “Phosphate buffer” to perform spray drying with reduced trehalose content or without trehalose. No dialysis was done with experiments with “Tris buffer” experiments. The spray drying parameters are shown in Table 5.Table 5. Spray drying parameters

[0199] Experiments were also performed with addition of silica (1.36% Silica Cab-o-sil) to both formulation buffers (Tris buffer and Phosphate buffer with high and low trehalose content) in order to investigate the effect on yield, flowability, and particle size.Table 6: Spray Drying Parameter with Silica

[0200] Next, the spray dried drug substance with the formulation buffer with and without silica addition were investigated for stability behavior with spray dried drug substance from. The materials were stored at controlled conditions (2-8°C, 25°C and 40°C with defined % relative humidity) for up to 6 months and subjected to a defined sample pull and analytical testing plan. Analytical testing included residual water content (Karl Fisher), analysis of crystallinity (WAXS), appearance, purity (SEC, IEX-HPLC and SDS- PAGE), particle size distribution (PSD), FT4 Flowability (shear cell) and potency.Table 7. Results of the spray drying studies

[0201] The results of the spray drying studies indicated no significant impact of a change in formulation (reduction of trehalose amount or change in buffer salts and reduction of trehalose as compared to standard condition) on the stability behavior of Kuma062 at 2- 8°C and 25±2°C (humidity of 60±5%) storage. Results for analytical testing as indicated above gave comparable results and trending. Samples at 40 ± 2°C and (humidity of 75±5%) showed altered material properties, including activation and degradation of Kuma062 for all three formulation options, most probably due to an unsuitable packaging configuration and exposure to air and high humidity. These results were thus considered not applicable for concluding on the impact of the different formulations on the stability behavior of Kuma062.Conclusion of Spray Drying Experiments

[0202] Drug substance spray drying was performed either with Tris buffer and with the alternative phosphate buffer with 47% trehalose and reduced (25%) trehalose content. Extensive analysis revealed that the quality attributes of Kuma062 protein and powdercharacteristics of the alternative phosphate buffer with reduced trehalose 25% content have similar protein characteristics to the currently used formulation buffer.

[0203] Therefore, development continued with “Phosphate buffer” with reduced (25%) trehalose content. Silica (w / w 1.36% Cab-o-sil) was added to the spray dried drug substance to increase flowability suitable for drug product processing.***

[0204] It is to be appreciated that the Detailed Description section, and not the Summary and Abstract sections, is intended to be used to interpret the claims. The Summary and Abstract sections may set forth one or more but not all exemplary aspects of the present disclosure as contemplated by the inventor(s), and thus, are not intended to limit the present disclosure and the appended claims in any way.

[0205] The present disclosure has been described above with the aid of functional building blocks illustrating the implementation of specified functions and relationships thereof. The boundaries of these functional building blocks have been arbitrarily defined herein for the convenience of the description. Alternate boundaries can be defined so long as the specified functions and relationships thereof are appropriately performed.

[0206] The foregoing description of the specific aspects will so fully reveal the general nature of the disclosure that others can, by applying knowledge within the skill of the art, readily modify and / or adapt for various applications such specific aspects, without undue experimentation, without departing from the general concept of the present disclosure. Therefore, such adaptations and modifications are intended to be within the meaning and range of equivalents of the disclosed aspects, based on the teaching and guidance presented herein. It is to be understood that the phraseology or terminology herein is for the purpose of description and not of limitation, such that the terminology or phraseology of the present specification is to be interpreted by the skilled artisan in light of the teachings and guidance.

[0207] The breadth and scope of the present disclosure should not be limited by any of the above-described exemplary aspects, but should be defined only in accordance with the following claims and their equivalents.

[0208] The contents of all cited references (including literature references, U.S. or foreign patents or patent applications, and websites) that are cited throughout this application are hereby expressly incorporated by reference as if written herein in their entireties for anypurpose, as are the references cited therein. Where any inconsistencies arise, material literally disclosed herein controls.

Claims

WHAT IS CLAIMED IS:

1. A method of producing a Kuma polypeptide comprising heat treating a cell suspension comprising one or more host cells comprising the polypeptide in a cell suspension buffer.

2. The method of claim 1, wherein the cell suspension buffer comprises a divalent cation.

3. The method of claim 2, wherein the divalent cation is Ca2+, Mg2+, Be2+, Mn2+, Zn2+, Sr2+,Ba2+, Cu2+, or Fe2+, or any combination thereof.

4. The method of claim 2 or 3, wherein the divalent cation is a calcium salt.

5. The method of claim 4, wherein the cell suspension buffer comprises from about 1 mmol / L CaCh to about 10 mmol / L CaCh.

6. The method of claim 4 or 5, wherein the cell suspension buffer comprises about 3.4 mmol / L CaCh.

7. The method of any one of claims 1-6, wherein the heat treatment comprises heating the cell suspension to about 40°C to about 90°C for about 5 seconds to about 15 minutes.

8. The method of any one of claims 1-7, wherein the heat treatment comprises heating the cell suspension to about 65°C for 1 minute.

9. The method of any one of claims 1-8, wherein the heat treatment further comprises a cooling step.

10. The method of claim 9, wherein the cooling step comprises cooling the cell suspension to about 0°C, to about 5°C, to about 10°C, to about 15°C, or to about 20°C.

11. The method of claim 9 or 10, wherein the cooling step comprises cooling the cell suspension to about 10°C.

12. The method of any one of claims 1-11, wherein the cell suspension buffer has a pH of from about 7.2 to about 10.2.

13. The method of any one of claims 1-12, wherein the cell suspension buffer has a pH of about 9.2.

14. The method of any one of claims 1-13, wherein the cell suspension buffer comprises arginine.

15. The method of claim 14, wherein the cell suspension buffer comprises about 4.2 g / L arginine.

16. The method of any one of claims 1-15, wherein the host cells are lysed into a lysed cell mixture as a result of the heat treatment.

17. The method of any one of claims 1-16, further comprising subjecting the lysed cell mixture to a flocculation.

18. The method of any one of claims 1-17, further comprising purifying the lysed cell mixture by calcium precipitation, thereby generating a purification mixture.

19. The method of claim 18, wherein the calcium precipitation comprises adding a phosphate solution to the purification mixture to result in a final phosphate concentration of about 10 mmol / L to about 50 mmol / L.

20. The method of claim 18 or 19, wherein the final phosphate concentration of the purification mixture is about 35 mmol / L.

21. The method of any one of claims 18-20, wherein the calcium precipitation comprises adding (i) calcium acetate or (ii) a mix of calcium acetate and calcium hydroxide to the purification mixture to result in a final concentration of calcium salt from about 10 mmol / L to about 90 mmol / L.

22. The method of any one of claims 18-21, wherein the final concentration of calcium salt in the purification mixture is about 70 mmol / L.

23. The method of any one of claims 18-22, wherein the purification mixture has a pH of about 6.5 to about 9.

24. The method of any one of claims 18-23, wherein the calcium precipitation is conducted at a temperature of from about 0 °C to about 10 °C.

25. The method of claim 24, wherein the calcium precipitation is conducted at about 10 °C.

26. The method of any one of claims 1-25, wherein the Kuma polypeptide comprises Kuma062 or a variant thereof.

27. The method of any one of claims 1-26, wherein the Kuma polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

28. The method of any one of claims 1-27, further comprising centrifugation, filtration, ultrafiltration / diafiltration, formulation, sterile filtration, spray drying, packing, storage, changing the arginine buffer, concentrating the polypeptide, or any combination thereof.

29. The method of claim 28, comprising changing the arginine buffer to a formulation buffer selected from the group consisting of: a) 23.37 mM K2HPO4, 1.63 mM KH2PO4, and 70 mM NaCl; b) 25 mM Glycine and 70 mM NaCl; c) 27 mM sodium acetate, 3 mM Taurin, and 70 mM NaCl;d) 27 mM sodium acetate, 3 mM Glycylglycine, and 70 mM NaCl; e) 27 mM sodium acetate, 3 mM sodium citrate, and 70 mM NaCl; f) 13 mM Tris and 70 mM NaCl; g) 20 mM K2HPO4 and 20 mM Glycine; h) 20 mM K2HPO4, 20 mM Glycine, and 70 mM NaCl; i) 23.37 mM K2HPO4, 1.63 mM KH2PO4, 25 mM Glycine, and 70 mM NaCl; j) 28.04 mM K2HPO4, 1.96 mM KH2PO4, 30 mM Glycine, and 70 mM NaCl; k) 30 mM Glycine and 70 mM NaCl; l) 50 mM Glycine and 70 mM NaCl; m) 45 mM sodium acetate, 5 mM Glycylglycine, and 70 mM NaCl; n) 25 mM sodium acetate;, 5 mM Glycylglycine, and 70 mM NaCl; o) 20 mM sodium acetate, 10 mM Glycylglycine, and 70 mM NaCl; p) 25 mM K2HPO4, lOmM Glycine, and 70 mM NaCl; q) 25 mM Na2HPO4, 5mM Glycine, and 70 mM NaCl; r) 20 mM sodium acetate, 10 mM Glycylglycine, and 70 mM NaCl; s) 13 mM Tris, 0.05% polysorbate 80, and 70 mM NaCl; t) 13 mM Tris and 70 mM NaCl; and u) 12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, and 70 mM NaCl.

30. The method of claim 29, wherein the formulation buffer further comprises from about 20% to about 50% trehalose.

31. The method of claim 30, wherein the formulation buffer further comprises about 25% trehalose.

32. The method of claim 31, wherein the formulation buffer further comprises about 47% trehalose.

33. The method of any one of claims 28-32, wherein the concentration of the Kuma polypeptide is from about 1 g / L to about 150 g / L.

34. The method of any one of claims 28-33, wherein the one or more processing steps comprises spray drying.

35. The method of claim 34, wherein the spray-drying solution comprises silica.

36. The method of claim 34, wherein the spray-drying solution does not comprise silica.

37. A composition comprising a Kuma polypeptide and a formulation buffer, wherein the formulation buffer is selected from the group consisting of: a) 23.37 mM K2HPO4, 1.63 mM KH2PO4, and 70 mM NaCl; b) 25 mM Glycine and 70 mM NaCl; c) 27 mM sodium acetate, 3 mM Taurin, and 70 mM NaCl; d) 27 mM sodium acetate, 3 mM Glycylglycine, and 70 mM NaCl; e) 27 mM sodium acetate, 3 mM sodium citrate, and 70 mM NaCl; f) 13 mM Tris and 70 mM NaCl; g) 20 mM K2HPO4 and 20 mM Glycine; h) 20 mM K2HPO4, 20 mM Glycine, and 70 mM NaCl; i) 23.37 mM K2HPO4, 1.63 mM KH2PO4, 25 mM Glycine, and 70 mM NaCl; j) 28.04 mM K2HPO4, 1.96 mM KH2PO4, 30 mM Glycine, and 70 mM NaCl; k) 30 mM Glycine and 70 mM NaCl; l) 50 mM Glycine and 70 mM NaCl; m) 45 mM sodium acetate, 5 mM Glycylglycine, 70 mM NaCl; n) 25 mM sodium acetate, 5 mM Glycylglycine, and 70 mM NaCl; o) 20 mM sodium acetate, 10 mM Glycylglycine, and 70 mM NaCl; p) 25 mM K2HPO4, lOmM Glycine, and 70 mM NaCl; q) 25 mM Na2HPO4, 5mM Glycine, and 70 mM NaCl; r) 20 mM sodium acetate, 10 mM Glycylglycine, and 70 mM NaCl;s) 13 mM Tris, 0.05% polysorbate 80 and 70 mM NaCl; t) 13 mM Tris and 70 mM NaCl; and u) 12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, and 70 mMNaCl.

38. The composition of claim 37, wherein the formulation buffer does not comprise trehalose.

39. The composition of claim 37 or 38, wherein the formulation buffer further comprises from about 20% to about 50% trehalose.

40. The composition of any one of claims 37-39, comprising 25% trehalose.

41. The composition of any one of claims 37-39, comprising 47% trehalose.

42. The composition of any one of claims 37-41, further comprising silica.

43. The composition of any one of claims 37-41, wherein the composition does not comprise silica.

44. The composition of any one of claims 37-43, wherein the concentration of the Kuma polypeptide is from about 1 g / L to about 150 g / L.

45. The composition of any one of claims 37-44, wherein the concentration of the Kuma polypeptide is about 100 g / L.

46. The composition of any one of claims 37-45, wherein the concentration of the Kuma polypeptide is about 2 g / L.

47. A process for producing a dry powder drug substance comprising a Kuma polypeptide comprising:a) suspending one or more host cells comprising the Kuma polypeptide in a cell suspension buffer; b) heat treating the cell suspension; c) flocculating the cell suspension; d) purifying the Kuma polypeptide by calcium precipitation; e) concentrating the Kuma polypeptide into a solution; f) changing the buffer in the concentrated solution to a formulation buffer, thereby producing a spray-drying solution comprising the Kuma polypeptide solution in the formuation buffer; and g) spray-drying the spray-drying solution comprising the Kuma polypeptide.

48. The process of claim 47, wherein the cell suspension comprises Ca2+.

49. The process of claim 47 or 48, wherein the heat treatment comprises heating the cell suspension at about 65°C for about 1 minute.

50. The process of any one of claims 47-49, wherein the heat treatment further comprises a cooling step, wherein the cooling step comprises cooling the cell suspension to about 0°C, to about 5°C, to about 10°C, or to about 15°C.

51. The process of any one of claims 47-50, wherein the cell suspension has a pH of about 9.

452. The process of any one of claims 47-51, wherein the phosphate-glycine buffer comprises 12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, and 70 mM NaCl.

53. The process of any one of claims 47-52, wherein the formulation buffer further comprises 25% or 47% trehalose.

54. The process of any one of claims 47-53, wherein the formulation buffer further comprises silica.

55. The process of any one of claims 47-53, wherein the formulation buffer does not comprise silica.

56. The process of any one of claims 47-55, wherein the Kuma polypeptide comprises Kuma062 or a variant thereof.

57. The process of any one of claims 47-56, wherein the Kuma polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

58. A composition comprising (i) a Kuma polypeptide and (ii) a formulation buffer comprising 12.5 mM K2HPO4, 12.5 mM Na2HPO4, 10 mM Glycine, and 70 mM NaCl.

59. The composition of claim 58, further comprising 25% trehalose.

60. The composition of claim 58, further comprising 47% trehalose.

61. The composition of any one of claims 58-60, further comprising silica.

62. The composition of any one of claims 58-61, wherein the Kuma polypeptide comprisesKuma062 or a variant thereof.

63. The composition of any one of claims 58-62, wherein the Kuma polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO: 1.

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