Method for purifying charge-shielded fusion proteins

Abstract

The present invention relates to a method for purifying charge-shielded proteins from cell lysates or periplasmic releases using hydrophobic interaction chromatography as a first chromatographic step. Compositions comprising the charge-shielded proteins and therapeutic methods using the purified charge-shielded proteins are also provided herein. TIFF2024500974000011.tif58170

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Application No. 63 / 130,295, filed December 23, 2020, the contents of which are incorporated in their entirety.

[0002] Submitting a sequence listing as an ASCII text file The contents of the following submission in an ASCII text file are incorporated herein in their entirety: Computer Readable Form (CRF) of Sequence Listing (Filename: 210462000140SEQLIST.TXT, Recorded on: December 9, 2021, Size: 30,160 bytes).

[0003] Field The present invention relates to a method for purifying a charge-shielded fusion protein. [Background technology]

[0004] background Many proteins of pharmaceutical interest, especially certain enzymes and recombinant antibody fragments, hormones, interferons, etc., undergo rapid (blood) clearance. This is especially true for proteins whose size is below the renal filtration threshold of about 70 kDa (Caliceti (2003) Adv Drug Deliv Rev 55:1261-1277). In these cases, the plasma half-life of the unmodified pharmaceutical protein may be of the order of a few hours, thus rendering it essentially useless for most therapeutic applications. In order to achieve sustained pharmacological action and improved patient compliance - the required dosing intervals extending up to days or even weeks - several strategies have been established in the past for the purpose of biopharmaceutical development.

[0005] One methodology for extending the plasma half-life of biopharmaceuticals is conjugation with highly solvated, physiologically inert chemical polymers, thus effectively extending the hydrodynamic radius of the therapeutic protein beyond the glomerular pore size of approximately 3-5 nm (Caliceti (2003) (Non-Patent Document 1)). Therefore, fusion proteins have been developed that contain a bioactive domain and an additional domain that increases the hydrophobic radius of the fusion protein without affecting the biological activity of the bioactive domain.

[0006] However, the production and purification of such fusion proteins presents challenges that require new purification methods. In particular, prior to the present invention, it was not known that the fusion of domains to increase the hydrodynamic radius of the bioactive domains can cause a charge shielding effect, thus making traditional purification methods inappropriate for such fusion proteins. The inventors have identified a charge shielding effect and a novel method for purifying such therapeutic fusion proteins. [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Caliceti (2003)Adv Drug Deliv Rev 55:1261-1277 Summary of the Invention

[0008] Quick Overview Provided herein is a method for purifying charge-shielded proteins from cell lysates or periplasmic release. In some embodiments, the method comprises hydrophobic interaction chromatography as a first chromatography step. In some embodiments, the method comprises anion exchange chromatography as a second chromatography step. In some embodiments, the method comprises cation exchange chromatography as a third chromatography step.

[0009] In some embodiments, provided herein is a method for purifying a charge-shielded fusion protein from a cell lysate or periplasmic release, the charge-shielded fusion protein comprising a biologically active domain and a charge-shielded domain, the method comprising hydrophobic interaction chromatography as a first chromatography step.

[0010] In some embodiments, provided herein is a method for producing a charge-shielded fusion protein from a cell lysate or periplasmic release, the charge-shielded fusion protein comprising a biologically active domain and a charge-shielded domain, the method comprising: i) culturing a cell comprising a nucleic acid encoding the charge-shielded fusion protein; and ii) purifying the charge-shielded fusion protein, the charge-shielded protein being purified from the cell lysate or periplasmic release using hydrophobic interaction chromatography as a first chromatography step.

[0011] In some embodiments, the charge-shielded fusion protein is at least 45% pure after the first chromatography step. In some embodiments, the method further comprises anion exchange chromatography. In some embodiments, the method further comprises cation exchange chromatography.

[0012] In some embodiments, the method comprises a series of chromatography steps comprising, in order, i) hydrophobic interaction chromatography, ii) anion exchange chromatography, and iii) cation exchange chromatography.

[0013] In some embodiments, the bioactive domain is charged at a pH of about 7.0, the charge shielding domain increases the hydrodynamic radius of the protein, and the charge shielding domain is uncharged at a pH of about 7.0. In some embodiments, the molecular weight of the bioactive domain is less than the molecular weight of the charge shielding domain. In some embodiments, the molecular weight of the charge shielding domain is between 10 kDa and 60 kDa. In some embodiments, the molecular weight of the charge shielding domain is between 10 kDa and 20 kDa. In some embodiments, the molecular weight of the bioactive domain is between 30 kDa and 40 kDa. In some embodiments, the molecular weight of the charge shielding domain is sufficient to increase the in vivo half-life of the charge shielded fusion protein or multimer of the charge shielded fusion protein. In some embodiments, the in vivo half-life of the charge shielded fusion or multimer of the charge shielded protein is increased compared to the half-life of a protein comprising the bioactive domain or multimer of a protein comprising the bioactive domain without a charge shielding domain.

[0014] In some embodiments, the charge shielding domain has a random coil or disordered structure. In some embodiments, the charge shielding domain is a polypeptide consisting of one or more of alanine, serine, and proline residues. In some embodiments, the charge shielding domain is a polypeptide consisting of proline and alanine residues.

[0015] In some embodiments, a method comprises purifying a PASylated bioactive fusion protein from a cell lysate or periplasmic release, comprising i) culturing a cell comprising a nucleic acid encoding a PASylated bioactive protein; and ii) purifying the PASylated bioactive protein, wherein the PASylated bioactive protein is purified from the cell lysate or periplasmic release using hydrophobic interaction chromatography as a first chromatography step.

[0016] In some embodiments, provided herein is a method for purifying a charge-shielded fusion protein comprising a biologically-active domain and a charge-shielded domain from a cell lysate or periplasmic release, the method comprising, in order, the steps of: i) applying a loading solution comprising the charge-shielded fusion protein to a hydrophobic interaction chromatography column; ii) applying a wash solution to the hydrophobic interaction chromatography column; iii) applying an elution solution to the hydrophobic interaction column to elute the charge-shielded protein; iv) applying the eluted charge-shielded fusion protein in iii) as a loading solution to an anion exchange chromatography column; v) eluting the charge-shielded fusion protein from the anion exchange chromatography column; vi) applying the eluted charge-shielded fusion protein in vi) as a loading solution to a cation exchange chromatography column; vii) applying a wash solution to the cation exchange chromatography column; and viii) applying an elution solution to the cation exchange chromatography column to elute the charge-shielded fusion protein.

[0017] In some embodiments, the loading solution in step i) comprises 2-3 M NaCl and has a pH of 6.0-8.0. In some embodiments, the elution solution in step iii) comprises 0.75-1.75 M NaCl and has a pH of 6.0-7.0. In some embodiments, the loading solution in step iv) has a conductivity of 0.7-4.0 mS / cm and a pH of 7.0 and 9.0. In some embodiments, the loading solution in step iv) has a conductivity of 0.7-4.0 mS / cm and a pH of 7.0 and 9.1. In some embodiments, the loading solution in step vi) has a pH of 6.0-7.0 and a conductivity of 0.7-2.5 mS / cm. In some embodiments, the loading solution in step vi) has a pH of 5.9-7.0 and a conductivity of 0.7-2.5 mS / cm.

[0018] In some embodiments, the elution solution in step viii) has a pH of 6.0-7.0 and a conductivity of 0.7-4.0 mS / cm. In some embodiments, the loading solution in step i) comprises 0.25-3 M Na2SO4 or 0.25-0.6 M NH4SO4 and a pH of 5.5-6.5. 20. In some embodiments, the elution solution in step iii) comprises 0.3-0.5 M NH4SO4 and has a pH of 5.5-6.5.

[0019] In some embodiments, the hydrophobic interaction chromatography is selected from the group consisting of POROS Benzyl Ultra resin, Hexyl-650C resin, and Phenyl-600M resin. In some embodiments, the hydrophobic interaction chromatography is Phenyl-600M resin. In some embodiments, the anion exchange interaction chromatography is selected from the group consisting of POROS 50HQ resin, POROS XQ resin, and Gigacap Q-650M resin. In some embodiments, the anion exchange interaction chromatography is Gigacap Q-650M resin. In some embodiments, the cation exchange interaction chromatography is a strong cation exchanger. In some embodiments, the cation exchange interaction chromatography is a mixed mode resin, the cation exchange interaction chromatography is selected from the group consisting of Capto MMC resin, CMM Hypercel resin, Capto SP impres resin, Fracto gel SO3-resin, GigaCap S-650S resin, and POROS XS resin. In some embodiments, the cation exchange interaction chromatography is POROS XS resin.

[0020] In some embodiments, the bioactive domain is an asparaginase subunit. In some embodiments, the asparaginase is selected from the group consisting of E. coli asparaginase and Erwinia asparaginase. In some embodiments, the asparaginase subunit comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7.

[0021] In some embodiments, the charge shielded fusion protein comprises the amino acid sequence shown in SEQ ID NO:9 or SEQ ID NO:10.

[0022] In some embodiments, the cell is a bacterial cell, hi some embodiments, the cell is an E. coli cell or a Pseudomonas cell.

[0023] Also provided herein are charge-shielded proteins produced by the methods provided herein.

[0024] In some embodiments, provided herein are compositions comprising a charge shielded protein and a pharma- ceutically acceptable carrier.

[0025] In some embodiments, provided herein are methods of treatment comprising administering to an individual in need of treatment a composition comprising a charge shielded protein, or a pharmaceutical composition comprising a charge shielded protein.

[0026] Also provided herein are compositions comprising a PAS-modified asparaginase, wherein the PAS-modified asparaginase is at least 45% pure. [Brief description of the drawings]

[0027] [Figure 1] 1 shows an SDS-PAGE of the elution from a POROS HQ anion exchange column as an initial protein capture step. The arrow indicates the band corresponding to PF745. [Diagram 2] Shown is an SDS-PAGE of the elution from a POROS XS cation exchange column as an initial protein capture step. [Diagram 3] SDS-CGE images of fractions from a representative Butyl-650M chromatography step are shown. For purity analysis, equal volumes (4 μL) of the load, flow-through (FT) from the load, elution, and stripping fractions were loaded onto SDS-CGE. The arrow indicates the band corresponding to PF745. [Figure 4] An overlaid comparison of representative POROS HQ chromatograms from three runs is shown. The chromatograms show volume in mL on the X-axis, absorbance at 280 nm in mAU on the left Y-axis, and conductivity in mS / cm on the right Y-axis. [Diagram 5] Shown are SDS-CGE images of fractions from the POROS XS chromatography step. For purity analysis, equal volumes (4 μL) of the load, flow-through from load (FT), wash, elution, strip 1 and strip 2 fractions were loaded onto CGE. The arrow indicates the band corresponding to PF745. [Figure 6] Shown is SDS-CGE of flow-through from a highly hydrophobic plate of HIC resin. Triplicate columns represent flow-through from wells spiked with kosmotrope concentrations indicated as A, B, C, or D. "A" was 0.25 M sodium sulfate, "B" was 0.5 M ammonium sulfate, "C" was 2 M NaCl, and "D" was 3 M NaCl. The MW of the expected target band (PF745) is indicated by an arrow to the left of the graph. [Figure 7] SDS-CGE of eluates from highly hydrophobic plates of HIC resin. Triplicate columns represent elution from wells spiked with kosmotrope concentrations indicated as A, B, C, or D. "A" was 0.25 M sodium sulfate, "B" was 0.5 M ammonium sulfate, "C" was 2 M NaCl, and "D" was 3 M NaCl. The MW of the expected PF745 band is indicated by an arrow to the left of the graph. [Figure 8] Shown is SDS-CGE of the flow-through from a low hydrophobicity plate of HIC resin. [Figure 9] 1 shows an SDS-CGE image of the elution of HIC resin from a low hydrophobicity plate. [Figure 10] SDS-CGE images of Phenyl-600M and Benzylultra chromatography are shown, demonstrating enrichment of PF745 in fractions 1B2-1C4 on Phenyl-600M and in fractions 2A5-2C3 on Benzylultra. [Figure 11] SDS-CGE images from anion exchange resin screening are shown. Target (PF745) purity is indicated above the main band in each lane. [Figure 12] 1 shows the SDS-CGE purity of flow-through fractions from POROS 50 HQ, POROS XQ, and GigaCap Q-650M chromatography runs. [Figure 13] A representative chromatogram of AEX using the GigaCap Q-650M is shown. [Figure 14] Shown are SDS-CGE images of flow-through fractions (in triplicate lanes) from a mixed-mode cation exchange resin, the top panel run at pH 5.7 and the bottom panel run at pH 6.0. [Figure 15] Shown are SDS-CGE images of Capto Core 400 load and flow-through fractions at various pH and salt concentrations as indicated above each lane. The % purity is indicated above each lane. [Figure 16] Shown are SDS-CGE images of NH2-750F load and flow-through fractions at various pH and conductivity indicated above each lane. The % purity is indicated above each lane. [Figure 17] Shown are SDS-CGE images of CaPure-HA fractions: load, flow-through (FT), wash, and elution, with the binding conditions indicated in the top lane of each set. [Figure 18] Shown are SDS-CGE images of PPG-600M fractions: load, flow-through (FT), wash, and elution, with the binding conditions indicated in the top lane of each set. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] Detailed Description I. Methods for Purifying Charge-Shielded Proteins In some embodiments, the methods provided herein include purifying the charge-shielded protein using one or more chromatography steps, and in some embodiments, the method includes hydrophobic interaction chromatography (HIC) as a first chromatography step. As used herein, the term "chromatography" includes methods of separating a mixture (e.g., a mixture of proteins in a cell lysate or periplasmic release). In some embodiments, chromatography includes separating a mixture, such as a cell lysate or periplasmic release, in a solution (e.g., a loading solution, a mobile phase) by passing the mixture through a medium that is on a stationary material (e.g., a resin, a stationary phase). The solution in a chromatography system can be configured as a liquid (e.g., liquid chromatography) or a vapor (e.g., gas chromatography). In some embodiments, chromatography separates a mixture in a solution through a medium that is on a stationary material, and the components of the mixture move at different speeds that cause them to separate from each other.

[0029] The composition of a particular loading solution and / or resin may determine the rate at which components of a mixture migrate, for example, certain components of a mixture may migrate slower through the resin (e.g., longer retention times) when a particular loading solution and / or resin is used, while other components of the same mixture may migrate faster through the resin (e.g., shorter retention times).

[0030] In some embodiments, the chromatographic separation of the mixture further comprises a resin (e.g., stationary phase), a loading solution (e.g., buffer, mobile phase), and a column. The composition of the resin and buffer may depend on and be specific to the particular chromatographic method described herein. In some embodiments, the chromatographic column contains the resin and allows the loading solution containing the mixture to pass through, being separated by chromatography. In some embodiments, the column is a glass, borosilicate glass, acrylic glass, or stainless steel chromatographic column.

[0031] In some embodiments, the methods provided herein relate to a capture purification step in which a cell lysate or periplasmic release is applied to a hydrophobic interaction chromatography column. As used herein, a "cell lysate" includes the contents of a lysed cell. As used herein, a "periplasmic release" includes the contents of the periplasm produced by lysis of the outer membrane. In some embodiments, the periplasmic release is a subfraction of the cell lysate. In some embodiments, the cell lysate or periplasmic release includes charge-shielded proteins expressed within the cell. Lysed cells can be obtained by breaking down the membrane of the cell, often by viral, enzymatic, or osmotic mechanisms to disrupt the integrity of the cell membrane. In some embodiments, cells are lysed by physical disruption, including but not limited to sonication, mechanical techniques (e.g., Waring blender polytron), liquid homogenization (e.g., using a Dance homogenizer, Potter-Elvehjem homogenizer, microfluidizer, or French press), freeze-thawing, and manual grinding (e.g., with a mortar and pestle). In alternative embodiments, cells are lysed by solution-based lysis, in which the cells are contacted with a cell lysis buffer that breaks the cells open and releases the contents of the cell. For example, cells can be lysed using a solution of buffered salts (e.g., Tris-HCl or MES) and ionic salts (e.g., NaCl or KCl). In some embodiments, additional components can be added to the cell lysis buffer, including protease inhibitors and detergents such as Triton X-100 or SDS, to prevent degradation of proteins released from the cells. In some embodiments, cell lysates or periplasmic releases are produced using any technique known in the art.

[0032] In some embodiments, the periplasmic release is produced by selectively disrupting the bacterial outer membrane. Methods for disrupting the bacterial outer membrane are known in the art. (See Wurm et al. Engineering in Life Sciences 17:215-222 (2017)). For example, treatments using guanidine HCl and / or Triton, cernitrate, benzalkonium chloride, glycerol ether, chloroform, TRIS, 1% glycine, polyethyleneimine, urea and DTT, mild heat shot and TRIS, and osmotic shock can all be used. In some embodiments, the outer membrane is disrupted during culture. In some embodiments, the outer membrane is disrupted after harvesting.

[0033] In some embodiments, the soluble fraction of the cell lysate or periplasmic release comprising the charge-shielded protein is separated from the insoluble fraction of the cell lysate or periplasmic release using centrifugation after lysis of the cell or extracellular membrane and prior to the first chromatographic capture step. In some embodiments, the soluble fraction of the cell lysate or periplasmic release is separated from the insoluble fraction of the cell lysate or periplasmic release by centrifugation. In some embodiments, the cell lysate or periplasmic release is centrifuged at up to about 3,000 x g, about 3,500 x g, about 4,000 x g, about 4,500 x g, about 5,000 x g, about 5,500 x g, about 6,000 x g, about 6,500 x g, about 7,000 x g, about 8,000 x g, about 9,000 x g, about 10,000 x g, about 11,000 x g, or about 15,000 x g, or more. In some embodiments, the cell lysate or periplasmic release is centrifuged at about 8,000-20,000 x g, about 5,000-6,000 x g, about 8,000-15,000 x g, about 18,000 x g, or about 20,000 x g. In some embodiments, the cell lysate or periplasmic release is centrifuged for up to about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 20 minutes, or about 30 minutes or more. In some embodiments, the cell lysate or periplasmic release is centrifuged for about 5-30 minutes, about 5-20 minutes, about 8-12 minutes, about 10-20 minutes, or about 15-30 minutes. Centrifugation may be performed at up to about 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C or more. In some embodiments, centrifugation is performed at about 0-10°C, or about 2-8°C.

[0034] In some embodiments, after centrifugation, the cell lysate or periplasmic release is subjected to one or more filtration or clarification steps prior to the first capture chromatography step. In some embodiments, the cell lysate or periplasmic release is subjected to ultrafiltration. In some embodiments, 0.2, 0.3, 0.4, 0.45, or 0.5 μm filters are used. In some embodiments, the cell lysate or periplasmic release is subjected to dialysis. In some embodiments, a buffer exchange is performed such that the cell lysate or periplasmic release is in a buffer suitable for application to the first hydrophobic interaction chromatography column.

[0035] In some embodiments, the soluble fraction of the cell lysate or periplasmic release isolated by centrifugation, which contains the charge-shielded protein, is applied to a capture step. As used herein, a "capture step" includes a first chromatographic step that binds the protein of interest (e.g., the charge-shielded protein) from the cell lysate. In some embodiments, the first chromatographic capture step isolates the protein of interest from whole cell lysate cellular contaminants, including, but not limited to, proteases and glycosidases, in addition to non-target host cell proteins. In some embodiments, the first chromatographic capture step enriches the target protein and preserves target protein activity. In some embodiments, the first chromatographic capture step can be optimized to maximize purification of the target protein from cellular contaminants (e.g., non-target host cell proteins). In some embodiments, prior to the first chromatographic capture step, the charge-shielded protein in the cell lysate is about 5%, about 6%, about 7%, about 8%, about 9%, or about 10% pure. In some embodiments, the charge-shielded fusion protein in the cell lysate is about 5-10%, about 6-8%, or about 7-9% pure prior to the first chromatographic capture step. In some embodiments, the charge-shielded fusion protein in the cell lysate is about 5-30%, about 10-30%, or about 15-20% pure prior to the first chromatographic capture step.

[0036] In some embodiments, the soluble fraction of the periplasmic release is applied to a capture step. In some embodiments, a chromatography step binds the protein of interest (e.g., the charge-shielded protein) from the periplasmic release. In some embodiments, the first chromatography capture step enriches the target protein and preserves the target protein activity. In some embodiments, the first chromatography capture step can be optimized to maximize purification of the target protein from cellular contaminants (e.g., non-target host cell proteins) present in the periplasmic release. In some embodiments, prior to the first chromatography capture step, the charge-shielded protein in the periplasmic release is about 5%, about 6%, about 7%, about 8%, about 9%, about 15%, about 18%, about 20%, about 25%, or about 30% pure. Pure. In some embodiments, prior to the first chromatography capture step, the charge-shielded fusion protein in the periplasmic release is about 5-30%, about 10-30%, or about 15-20% pure.

[0037] The methods described herein may include purifying the charge-shielded fusion protein using chromatography (e.g., from cell lysates or periplasmic release). In some embodiments, the methods described herein may include purifying the charge-shielded fusion protein using multiple chromatography steps. In some embodiments, the method for purifying the charge-shielded fusion protein includes 1, 2, 3, 4, 5, 6, or 7 chromatography steps. In some embodiments, the method for purifying the charge-shielded fusion protein includes 1-7, or 1-3, or 3-5 chromatography steps. The chromatography may include liquid chromatography or gas chromatography. In some embodiments, the method includes HIC, anion exchange (AEX) chromatography, cation exchange (CEX) chromatography, ion exchange (IEX) chromatography, partition chromatography, normal phase chromatography, displacement chromatography, reversed phase chromatography (RPC), bioaffinity chromatography, aqueous normal phase chromatography, high performance liquid chromatography, flash chromatography, or other chromatographic methods.

[0038] In some embodiments, the charge-shielded fusion protein has a purity of about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% after the first chromatographic capture step. In some embodiments, the charge-shielded fusion protein has a purity of about 50%-80%, or about 60%-80% after the first chromatographic capture step. In some embodiments, the charge-shielded fusion protein has a purity of at least 45% after the first chromatographic capture step. In some embodiments, the purity of the charge-shielded protein exceeds the purity of the charge-shielded protein after the first HIC chromatography step compared to the purity of the charge-shielded protein using an ion exchange chromatography step. In some embodiments, the purity of the charge-shielded protein exceeds the purity of the charge-shielded protein after the first HIC chromatography step when purified according to the method of the bioactive domain.

[0039] The charge-shielded fusion protein may have increased purity when the first chromatography step is combined with the second chromatography step compared to a single chromatography step. The charge-shielded fusion protein may have increased purity when the first chromatography step is combined with the second and third chromatography steps compared to a single chromatography step. The charge-shielded fusion protein may have increased purity when the first and second chromatography steps are combined with the third chromatography step compared to two chromatography steps.

[0040] In some embodiments, the method includes a first chromatography, or capture step (e.g., HIC). In some embodiments, the first HIC step is followed by a second HIC step. In some embodiments, the first HIC step is followed by an AEX chromatography step. Alternatively, the first HIC step can be followed by a CEX chromatography step. In some embodiments, the first HIC step is followed by a chromatography step consisting of any chromatography technique described herein or otherwise known to the skilled artisan. The second chromatography step following the first HIC step is optionally followed by a third chromatography step. In one aspect, the third chromatography step is a CEX chromatography step. In another aspect, the third chromatography step is an AEX chromatography step. In some embodiments, a CEX chromatography step is performed after the first HIC step and the AEX chromatography step. In some embodiments, an AEX chromatography step is performed after the first HIC step and the CEX chromatography step. In an alternative method, the third chromatography step, consisting of any chromatography technique described herein or otherwise known to one of skill in the art, is performed after the first HIC step and the AEX step. Further embodiments include a third chromatography step, consisting of any chromatography technique described herein or otherwise known to one of skill in the art, performed after the first HIC step and the CEX step.

[0041] Between each chromatography step, one or more optional ultrafiltration (UF) and / or diafiltration (DF) (e.g., UF / DF) steps may be performed. In some embodiments, UF / DF is performed for concentration and buffer exchange between chromatography steps. For example, UF / DF may include separation by filtration. In some embodiments, the eluate from the chromatography step is contacted with the membrane under applied pressure. In some embodiments, this applied pressure drives the migration of the elution solution, buffer salts, and smaller non-target solution components through the membrane. In some embodiments, the membrane retains larger molecules (e.g., target proteins).

[0042] In some embodiments, the methods provided herein include using HIC as a first chromatographic step. In some embodiments, HIC includes methods for separating mixtures based on their hydrophobicity. HIC can include applying a mixture including a buffer and a protein, including both hydrophilic and hydrophobic regions, to a HIC resin in a chromatographic column. In some embodiments, HIC is performed as a first chromatographic step using a HIC-specific resin. In some embodiments, the HIC resin is a highly hydrophobic HIC resin. In some embodiments, the HIC resin is a low hydrophobic resin. In some embodiments, the purity of the composition including the charge-shielded fusion protein is about 40%, about 50%, about 60%, about 70%, about 80%, about 85%, about 90%, or about 95% after the HIC capture chromatography step. In some embodiments, the charge-shielded fusion protein has a purity of about 50%-80%, or about 60%-80%, or about 80%-95% after the HIC capture chromatography step. In some embodiments, the charge-shielded fusion protein has a purity of at least 45% after the HIC capture chromatography step.

[0043] In some embodiments, the HIC resin has a pore size of up to or greater than about 500 Å, about 550 Å, about 600 Å, about 650 Å, about 700 Å, about 750 Å, about 800 Å, about 850 Å, about 900 Å, about 950 Å, about 1,000 Å, about 1,500 Å, or about 2,000 Å. In some embodiments, the HIC resin has a pore size of about 500-2,000 Å, about 700-1,000 Å, about 700-800 Å, or about 900-1,500 Å. In some embodiments, the HIC resin has a particle size of at or above about 40 μm, about 45 μm, about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, about 100 μm, about 105 μm, about 110 μm, about 115 μm, or about 120 μm, In some embodiments, the HIC resin has a particle size of about 40-120 μm, about 60-100 μm, about 70-110 μm, and about 40-50 μm.

[0044] In some embodiments, the HIC resin is comprised of a matrix support substrate, the substrate being a hydrophilic carbohydrate. The HIC resin substrate can be cross-linked agarose or a synthetic copolymer material. In some embodiments, the HIC resin is comprised of a cross-linked poly[styrene-divinylbenzene] substrate or a hydroxylated methacrylate polymer substrate. In some embodiments, the HIC resin further comprises a ligand functional group attached to the substrate, the ligand functional group being hydrophobic. The HIC ligand functional group can be a linear alkyl ligand exhibiting hydrophobicity, or an aryl ligand exhibiting mixed mode behavior, in which both aromatic and hydrophobic interactions are possible. In some embodiments, the ligand functional group is an aromatic hydrophobic benzyl ligand, a phenyl ligand, or a C6 (hexyl) group. In some embodiments, the HIC resin is comprised of a cross-linked poly[styrene-divinylbenzene] substrate coupled with an aromatic hydrophobic benzyl ligand functional group. In some embodiments, the HIC resin is comprised of a hydroxylated methacrylate polymer substrate coupled with a C6 (hexyl) group. In some embodiments, the HIC resin is comprised of a hydroxylated methacrylate polymer substrate coupled with a phenyl functional group. In some embodiments, the HIC resin is POROS Benzyl ultra resin, POROS Benzyl resin, Capto Phenyl (high sub) resin, Butyl-650M resin, Hexyl-650C resin, Phenyl-600M resin, Capto Phenyl ImpRes resin, Phenyl Sepharose HP resin, Octyl Sepharose 4 FF resin, Capto Octyl resin, PPG-600M resin, or POROS Ethyl resin.

[0045] In many cases, the HIC resin may be equilibrated using an equilibration buffer prior to application of a loading solution containing the charge-shielded fusion protein. In some embodiments, the HIC equilibration buffer comprises a buffered salt solution. In some embodiments, the HIC equilibration buffer comprises Tris, EDTA, and a salt (e.g., NaCl). In some embodiments, the HIC equilibration buffer is equilibrated to a pH of about 5.0-10.0, or up to about pH 5.0, about pH 6.0, about pH 7.0, about pH 8.0, about pH 9.0, or about pH 10.0, or greater. In some embodiments, the HIC equilibration buffer is selected based on the use of the particular HIC resin in the first chromatographic capture step. Optionally, the HIC equilibration solution comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts.

[0046] In some embodiments, the charge-shielded fusion protein is applied to the HIC resin in a mixture, and the mixture comprising the charge-shielded fusion protein comprises a loading solution. In some embodiments, the loading solution comprising the charge-shielded fusion protein is applied to the HIC resin. In some embodiments, the loading solution comprises a salt solution. In some embodiments, the salt solution of the HIC loading solution comprises NaCl, (NH4)2SO4, Na2SO4, KCl, or CH3COONH4. In some embodiments, the salt solution of the HIC loading solution comprises about 1 M NaCl, about 2 M NaCl, about 3 M NaCl, about 4 M NaCl, or about 5 M NaCl. In some embodiments, the salt solution comprises about 1-5 M NaCl, or about 2-3 M NaCl. In some embodiments, the HIC loading solution comprising the charge shielded fusion protein loaded onto the HIC resin has a pH of about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0, or less than or greater than these. In some embodiments, the HIC loading solution comprising the charge shielded fusion protein loaded onto the HIC resin has a pH of about 5.0-9.0, or about 6.0-8.0. Optionally, the loading solution comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts.

[0047] In some embodiments, the loading solution comprises about 0.25 to about 3 M Na2SO4, such as about 0.4 to about 3.0 M Na2SO4, about 0.5 to about 3 M Na2SO4, about 0.4 to about 2 M Na2SO4, or about 0.4 to about 1.0 M Na2SO4. In some embodiments, the loading solution comprises about 0.6 M Na2SO4. In some embodiments, the loading solution has a pH of 5.5 to 6.5, such as pH 5.5 to 6.3, pH 5.6 to 6.3, or pH 5.7 to 6.2. In some embodiments, the loading solution has a pH of about pH 5.9. In some embodiments, the loading solution comprises about 0.25 to about 0.6 M NH4SO4, about 0.3 to about 0.6 M NH4SO4, or about 0.4 to about 0.6 M NH4SO4.

[0048] After applying the HIC loading solution containing the charge-shielded fusion protein to the HIC resin, one or more washing steps may be performed using a washing buffer. The washing buffer is selected based on the HIC loading solution and the particular HIC resin, and it will be apparent to one of skill in the art that a variety of washing buffers can be used. In some embodiments, the washing buffer comprises a salt solution. In some embodiments, the washing buffer comprises NaCl, (NH4)2SO4, Na2SO4, KCl, or CH3COONH4. In some embodiments, the washing buffer further comprises Tris and EDTA. Optionally, the washing buffer comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts. In some embodiments, the HIC washing buffer is the same as the HIC equilibration buffer. Alternatively, the HIC washing buffer may be different from the HIC equilibration buffer.

[0049] In some embodiments, the purified charge-shielded fusion protein is eluted from the HIC resin, optionally after one or more washes. The HIC elution solution comprises a salt solution. In some embodiments, the HIC elution solution salt solution is a NaCl buffer. In some embodiments, the NaCl buffer comprises about 0.6 M NaCl, about 0.65 M NaCl, about 0.7 M NaCl, about 0.75 M NaCl, about 0.8 M NaCl, about 0.85 M NaCl, about 0.9 M NaCl, about 1 M NaCl, about 1.2 M NaCl, about 1.5 M NaCl, about 1.75 M NaCl, about 2 M NaCl, or about 2.5 M NaCl. In some embodiments, the NaCl buffer comprises about 0.6-2.5 M NaCl or about 0.75-1.75 M NaCl. In some embodiments, the HIC elution solution has a pH of about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5. In some embodiments, the HIC elution solution has a pH of about 5.5-7.5, or about 6.0-7.0. Optionally, the elution solution includes additives, including but not limited to, detergents, alcohols, and chaotropic salts.

[0050] In some embodiments, the HIC elution solution comprises about 0.3 to about 0.5 M NH4SO4 and has a pH of about 5.5 to about 6.5. In some embodiments, the HIC elution solution comprises about 0.35 to about 0.45 M NH4SO4, or about 0.4 M NH4SO4. In some embodiments, the HIC elution solution has a pH of about pH 5.6 to about 6.4, about pH 5.7 to about 6.2, or about pH 5.9.

[0051] In some embodiments, HIC is carried out at about room temperature, hi some embodiments, HIC is carried out at about 15°C to about 28°C, or about 18°C ​​to about 25°C.

[0052] In some embodiments, the HIC is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. In some embodiments, the first HIC capture step is performed 1-15 times, 3-6 times, 8-10 times, or 9-15 times. Optionally, the eluate from the first HIC capture step can be stored at 0°C, about 1°C, about 2°C, about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, or about 10°C until ready for further processing. In some embodiments, the eluate from the first HIC capture step is stored at about 4°C to about 8°C. In some embodiments, the eluate from the first HIC capture step is stored at about 5-25°C, about 2-8°C, about 10-20°C, or about 18°C ​​to 25°C until ready for further processing. In some embodiments, the eluate is stored for up to about 8 hours at about 25° C. In some embodiments, the eluate is stored at about 4° C. to about 8° C. for more than 24 hours.

[0053] In some embodiments, the methods provided herein include purifying the charge-shielded fusion protein using one or more chromatography steps, and in some embodiments, the methods include AEX chromatography as a chromatography step following HIC. In some embodiments, the AEX chromatography step is the second chromatography step following the first HIC step. AEX chromatography is a process that uses IEX resins containing positively charged groups to separate substances based on their net surface charge. In solution, the resin is coated with positively charged counterions. Thus, the positively charged groups on the AEX resin bind to negatively charged proteins in solution. In some embodiments, the AEX resin used in the methods described herein is a strong anion exchange resin. In some embodiments, the AEX resin used in the methods described herein is a weak anion exchange resin. The classification of AEX resins as "strong" or "weak" anion exchangers refers to the extent to which the ionization state of the resin functional groups changes with pH. For example, a weak AEX resin is ionized over a limited pH range (e.g., the functional groups either pick up or lose protons with changes in buffer pH), whereas a strong AEX resin does not show a change in ion exchange capacity with changes in pH (e.g., the functional groups do not change and remain fully charged over a wide pH range).

[0054] In many cases, the AEX resin may be equilibrated using an equilibration buffer prior to application of the AEX loading solution containing the charge-shielded fusion protein. In some embodiments, the AEX equilibration buffer comprises a buffered salt solution. In some embodiments, the AEX equilibration buffer comprises Tris, EDTA, and a salt (e.g., NaCl). In some embodiments, the AEX equilibration buffer is equilibrated to a pH of about 5.0-10.0, or up to about pH 5.0, about pH 6.0, about pH 7.0, about pH 8.0, about pH 9.0, or about pH 10.0, or greater. In some embodiments, the AEX equilibration buffer is selected based on the use of the particular AEX resin in the second chromatography step. Optionally, the AEX equilibration solution comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts.

[0055] In some embodiments, the AEX resin has a pore size of up to about 500 Å, about 600 Å, about 700 Å, about 800 Å, about 900 Å, about 1,000 Å, about 2,000 Å, about 3,000 Å, about 4,000 Å, about 5,000 Å, about 6,000 Å, about 7,000 Å, about 8,000 Å, about 9,000 Å, or about 10,000 Å, or more. In some embodiments, the AEX resin has a pore size of about 500-10,000 Å, about 500-800 Å, about 900-1,200 Å, or about 5,000-10,000 Å. In some embodiments, the AEX resin has a particle size of up to about 50 μm, about 55 μm, about 60 μm, about 65 μm, about 70 μm, about 75 μm, about 80 μm, about 85 μm, about 90 μm, about 95 μm, or about 100 μm, or more, In some embodiments, the AEX resin has a particle size of about 50-100 μm, about 70-80 μm, about 50-90 μm, or about 80-100 μm.

[0056] In some embodiments, the AEX resin is composed of a poly[styrene-divinylbenzene] or hydroxylated methacrylic polymer substrate. The AEX resin substrate may be optionally coated with an additional polyhydroxyl surface coating to ensure low non-specific binding. In some embodiments, the AEX resin further comprises a ligand functional group attached to the substrate, the ligand functional group being positively charged or basic. The AEX ligand functional group may be a weak ion exchanger or a strong ion exchanger. For example, a weak AEX ligand functional group may include diethylaminoethyl or diethylaminopropyl. Alternatively, a strong AEX ligand functional group may include a quaternary ammonium or amine group. In some embodiments, the AEX resin is composed of a rigid, highly porous cross-linked poly[styrene-divinylbenzene] substrate with an additional polyhydroxyl surface coating to ensure low non-specific binding, coupled with a quaternized polyethyleneimine functional group. In some embodiments, the AEX resin is composed of a rigid, highly porous cross-linked poly[styrene-divinylbenzene] substrate with an additional polyhydroxyl surface coating to ensure low non-specific binding and coupled with a fully quaternized amine. In some embodiments, the AEX resin is composed of a hydroxylated methacrylic polymer substrate that has been chemically modified to provide more anion binding sites and coupled with a strong AEX functional group of quaternary amines. In some embodiments, the AEX resin is POROS 50HQ resin, POROS XQ resin, Gigacap Q-650M resin, Super Q-650M resin, or NH2-750F resin.

[0057] In some embodiments, the charge-shielded fusion protein is applied to the AEX resin in a mixture, and the mixture containing the charge-shielded fusion protein comprises a loading solution comprised of an eluate from the HIC step. In some embodiments, the loading solution containing the charge-shielded fusion protein is applied to the AEX resin. In some embodiments, the loading solution comprises a salt. In some embodiments, the AEX loading solution has a conductivity of about 0.5 mS / cm, about 0.6 mS / cm, about 0.7 mS / cm, about 0.8 mS / cm, about 0.9 mS / cm, about 1.0 mS / cm, about 2.0 mS / cm, about 3.0 mS / cm, about 4.0 mS / cm, about 5.0 mS / cm, and about 6.0 mS / cm, or less than or greater than these. In some embodiments, the AEX loading solution has a conductivity of about 0.5-6.0 mS / cm, or a conductivity of about 0.7-4.0 mS / cm. In some embodiments, the AEX loading solution has a pH less than or greater than about 6.0, about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, about 9.0, about 9.5, or about 10.0. In some embodiments, the AEX loading solution has a pH between about 6.0 and 10.0, or between about 7.0 and 9.0. In some embodiments, the AEX loading solution has a pH between 7.0 and 9.1.

[0058] After applying the AEX loading solution containing the charge-shielded fusion protein to the AEX resin, one or more washing steps may be performed using a washing buffer. The washing buffer is selected based on the AEX loading solution and the particular AEX resin. In some embodiments, the washing buffer comprises a salt solution. In some embodiments, the washing buffer comprises NaCl, (NH4)2SO4, Na2SO4, KCl, or CH3COONH4. In some embodiments, the washing buffer further comprises Tris and EDTA. Optionally, the washing buffer comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts. In some embodiments, the AEX washing buffer is the same as the AEX equilibration buffer. Alternatively, the AEX washing buffer may be different from the AEX equilibration buffer.

[0059] In some embodiments, the purified charge shielded fusion protein is applied to the AEX resin, and the flow-through is collected, optionally following one or more washes. In some embodiments, all of the AEX flow-through and all of the washes are collected. One or more second chromatography steps, optionally including AEX, may be performed to obtain sufficient material for subsequent downstream processing. In some embodiments, the AEX chromatography steps are performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. In some embodiments, the AEX chromatography steps are performed 1-15 times, 3-6 times, 8-10 times, or 9-15 times. Optionally, the flow-through from the AEX chromatography step may be stored at 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., or about 10° C. until ready for further processing. In some embodiments, the eluate from the AEX chromatography step is stored at about 0-10° C., or about 2-8° C. until ready for further processing. In some embodiments, the eluate from the AEX chromatography step is stored at about 4° C. to about 8° C. In some embodiments, the eluate from the AEX chromatography step is stored at about 5-25° C., about 2-8° C., about 10-20° C., or about 18° C. to 25° C. until ready for further processing. In some embodiments, the eluate is stored at about 25° C. for up to about 8 hours. In some embodiments, the eluate is stored at about 4° C. to about 8° C. for more than 24 hours.

[0060] In some embodiments, the methods provided herein include purifying the charge-shielded fusion protein using one or more chromatography steps, and in some embodiments, the methods include CEX chromatography as a chromatography step following HIC. In some embodiments, the CEX chromatography step is the second chromatography step following the first HIC step. In some embodiments, the CEX chromatography step is the third chromatography step following the first HIC step and the AEX chromatography step. CEX chromatography is a process of separating substances based on their net surface charge using IEX resins that contain negatively charged groups. In solution, the resins are coated with negatively charged counterions. Thus, the negatively charged groups on the CEX resins bind to positively charged proteins in solution. In some embodiments, the CEX resins used in the methods described herein are strong cation exchange resins. In some embodiments, the CEX resins used in the methods described herein are weak cation exchange resins. The classification of CEX resins as "strong" or "weak" anion exchangers refers to the extent to which the ionization state of the resin functional groups changes with pH. For example, a weak CEX resin is ionized over a limited pH range (e.g., the functional groups either pick up or lose protons with changes in buffer pH), whereas a strong CEX resin does not show a change in ion exchange capacity with changes in pH (e.g., the functional groups do not change and remain fully charged over a wide pH range).

[0061] In some embodiments, the CEX resin is a mixed-mode resin. Mixed-mode chromatography includes chromatographic methods that utilize two or more types of interactions between a stationary phase and a loading solution to achieve separation of target proteins. Most mixed-mode phases are typically silica or polymer reversed-phase based materials coupled with ion-exchange ligand functional groups. For example, a mixed-mode CEX resin may contain negatively charged sulfonate groups covalently attached to the reversed-phase backbone.

[0062] In many cases, the CEX resin may be equilibrated using an equilibration buffer prior to application of the CEX loading solution containing the charge-shielded fusion protein. In some embodiments, the CEX equilibration buffer comprises a buffered salt solution. In some embodiments, the CEX equilibration buffer comprises Tris, EDTA, and a salt (e.g., NaCl). In some embodiments, the CEX equilibration buffer is equilibrated to a pH of about 5.0-10.0, or up to about pH 5.0, about pH 6.0, about pH 7.0, about pH 8.0, about pH 9.0, or about pH 10.0, or greater. In some embodiments, the equilibration buffer is selected based on the use of the particular CEX resin in the second chromatography step. Optionally, the equilibration solution comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts.

[0063] In some embodiments, the CEX resin has a pore size of up to about 500 Å, about 600 Å, about 700 Å, about 800 Å, about 900 Å, about 1,000 Å, or about 2,000 Å, or more. In some embodiments, the CEX resin has a pore size of about 500-2,000 Å, about 800-1,000 Å, or about 700-900 Å. In some embodiments, the CEX resin has a particle size of up to about 10 μm, about 20 μm, about 30 μm, about 40 μm, about 50 μm, about 60 μm, about 70 μm, about 80 μm, about 90 μm, or about 100 μm, or more. In some embodiments, the CEX resin has a particle size of about 20-100 μm, about 30-50 μm, about 50-80 μm, or about 80-100 μm.

[0064] In some embodiments, the CEX resin is comprised of a poly[styrene-divinylbenzene], methacrylate polymer, agarose, or cellulose substrate. The CEX resin substrate may be coated with an additional polyhydroxyl surface coating to ensure low non-specific binding. In some embodiments, the CEX resin further comprises a ligand functional group attached to the substrate, the ligand functional group being negatively charged or acidic. The CEX ligand functional group may be a weak cation exchanger or a strong cation exchanger. For example, the weak CEX ligand functional group may comprise a carboxymethyl group. Alternatively, the strong CEX ligand functional group may comprise a sulfonic acid (e.g., methylsulfonate, sulfonyl, sulfoisobutyl, sulfopropyl), a carboxylic acid (e.g., carboxymethyl), or a phosphonic acid. In some embodiments, the CEX ligand functional group may comprise, in addition to the negatively charged CEX group, a multimodal (e.g., mixed mode) functional group that includes a primary amine, or a group that provides hydrogen bonding and hydrophobic interaction sites.

[0065] In some embodiments, the CEX resin is composed of a rigid, highly porous, cross-linked poly[styrene-divinylbenzene] substrate with an additional polyhydroxyl surface coating to ensure low non-specific binding, coupled with a high density of negatively charged sulfopropyl functional groups. In some embodiments, the CEX resin is composed of a rigid, high-flow agarose-based matrix coupled with a multimodal weak CEX ligand functional group containing carboxylic acid groups and additional groups that provide hydrogen bonding and hydrophobic interaction sites. In some embodiments, the CEX resin is composed of a rigid cellulose-based matrix coupled with a ligand containing both primary amine and carboxyl groups, conferring CEX and hydrophobic properties. In some embodiments, the CEX resin is composed of a high-flow agarose-based matrix coupled with negatively charged sulfonate (SP) groups. In some embodiments, the CEX resin is composed of a synthetic methacrylate polymer substrate coupled with negatively charged sulfoisobutyl functional ion exchanger groups via linear polymer chains. In some embodiments, the CEX resin is comprised of a high resolution, high capacity CEX resin that includes a methacrylate polymer substrate that has been chemically modified to provide a greater number of cationic binding sites and is coupled with a sulfopropyl (S) strong CEX functional group. In some embodiments, the CEX resin is Capto MMC resin, CMM Hypercel resin, Capto SP impres resin, Fracto gel SO3-resin, GigaCap S-650S resin, POROS XS resin, MX-TRP-650M resin, Sulfate-650F resin, NH2-750F resin, CaPure-HA resin, or PPG-600M resin.

[0066] In some embodiments, the charge-shielded fusion protein is applied to the CEX resin in a mixture, and the mixture containing the charge-shielded fusion protein comprises a loading solution consisting of an eluate from a previous chromatography step (e.g., AEX chromatography or HIC). In some embodiments, the loading solution containing the charge-shielded fusion protein is added to the CEX resin and comprises about a salt solution. In some embodiments, the CEX loading solution has a conductivity of about 0.5 mS / cm, about 0.6 mS / cm, about 0.7 mS / cm, about 0.8 mS / cm, about 0.9 mS / cm, about 1.0 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3.5 mS / cm, and about 4.0 mS / cm, or less than or greater than these. In some embodiments, the CEX loading solution has a conductivity of about 0.5-4.0 mS / cm, or a conductivity of about 0.7-2.5 mS / cm. In some embodiments, the CEX loading solution has a pH of about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0, or less than or greater than these. In some embodiments, the CEX loading solution has a pH of about 5.0-8.0, or about 6.0-7.0. In some embodiments, the CEX loading solution has a pH of 5.9-7.0.

[0067] After applying the CEX loading solution containing the charge-shielded fusion protein to the CEX resin, one or more washing steps may be performed using a washing buffer. The washing buffer is selected based on the CEX loading solution and the particular CEX resin, and it will be apparent to one skilled in the art that a variety of washing buffers can be used. In some embodiments, the washing buffer comprises a salt solution. In some embodiments, the washing buffer comprises NaCl, (NH4)2SO4, Na2SO4, KCl, or CH3COONH4. In some embodiments, the washing buffer further comprises Tris or MES and EDTA. Optionally, the washing buffer comprises additives, including, but not limited to, detergents, alcohols, and chaotropic salts. In some embodiments, the CEX washing buffer is the same as the CEX equilibration buffer. Alternatively, the CEX washing buffer may be different from the CEX equilibration buffer.

[0068] In some embodiments, the purified charge-shielded fusion protein is eluted from the CEX resin, optionally after one or more washes. The CEX elution solution comprises a salt solution. In some embodiments, the CEX elution solution has a conductivity of about 0.5 mS / cm, about 0.6 mS / cm, about 0.7 mS / cm, about 0.8 mS / cm, about 0.9 mS / cm, about 1.0 mS / cm, about 2.0 mS / cm, about 2.5 mS / cm, about 3.0 mS / cm, about 3 mS / cm, about 4.0 mS / cm, or about 5.0 mS / cm, or less than or greater than these. In some embodiments, the CEX elution solution has a conductivity of about 0.5-5.0 mS / cm, about 0.7-4.0 mS / cm, about 1.0-2.0 mS / cm, or about 3.0-4.0 mS / cm. In some embodiments, the CEX elution solution has a pH of about 5.5, about 6.0, about 6.5, about 7.0, or about 7.5. In some embodiments, the CEX elution solution has a pH of about 5.5-7.5, or a pH of about 6.0-7.0.

[0069] A third chromatography step, optionally including CEX, may be performed one or more times to obtain sufficient material for subsequent downstream processing. In some embodiments, the CEX chromatography step is performed 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 times. In some embodiments, the CEX chromatography step is performed 1-15 times, 3-6 times, 8-10 times, or 9-15 times. Optionally, the eluate from the CEX chromatography step may be stored at 0° C., about 1° C., about 2° C., about 3° C., about 4° C., about 5° C., about 6° C., about 7° C., about 8° C., about 9° C., or about 10° C. until ready for further processing. In some embodiments, the eluate from the AEX chromatography step is stored at about 0-10° C., or about 2-8° C. until ready for further processing.

[0070] II. Methods for Producing Charge-Shielded Fusion Proteins In some embodiments, the methods provided herein include culturing cells comprising a nucleic acid encoding a charge-shielded protein to produce a charge-shielded fusion protein and purifying the charge-shielded fusion protein. Host cells for expression of polypeptides are well known in the art and include prokaryotic cells as well as eukaryotic cells, such as E. coli cells, Pseudomonas fluorescens cells, yeast cells, invertebrate cells, CHO cells, CHO-K1 cells, Hela cells, COS-1 monkey cells, melanoma cells such as Bowes cells, mouse L-929 cells, 3T3 lines from Swiss, Balb-c or NIH mice, BHK or HaK hamster cell lines.

[0071] In some embodiments, the nucleic acid encoding the charge shielded protein is in a vector, hi some embodiments, the nucleic acid encoding the charge shielded protein is integrated into a host cell chromosome.

[0072] Preferably, the vector is an expression vector and / or a gene transfer or targeting vector. Expression vectors derived from viruses such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses, or bovine papilloma viruses can be used to deliver the polynucleotides or vectors of the invention to targeted cell populations. The vectors containing the nucleic acid molecules of the invention can be transferred into host cells by well-known methods, which vary depending on the type of cellular host.

[0073] Charge-shielded fusion proteins can be produced by recombinant DNA techniques, for example, by culturing cells containing the described nucleic acid molecules or vectors encoding the charge-shielded fusion proteins and isolating the biologically active protein from the culture. Charge-shielded fusion proteins can be produced in any suitable cell culture system, including prokaryotic cells, such as E. coli (e.g., BL21, W3110, or JM83), P. fluorescens, or Bacillus subtilus, or eukaryotic cells, such as Pichia pastoris yeast strain X-33 or CHO cells. Further suitable cell lines known in the art are available from cell line depositories, such as the American Type Culture Collection (ATCC). The term "prokaryotic" is meant to include bacterial cells, and the term "eukaryotic" is meant to include yeast, higher plant, insect, and mammalian cells. The transformed host can be grown in a fermenter and cultured according to techniques known in the art to achieve optimal cell growth. In a further embodiment, the present invention relates to a process for the preparation of said bioactive proteins comprising culturing the cells of the invention under conditions suitable for expression of the bioactive protein and isolating the bioactive protein from the cells or the culture medium.

[0074] Further examples of methods, vectors, and translation and transcription elements and other elements useful in the methods herein are described, for example, in U.S. Pat. No. 5,055,294 by Gilroy, and U.S. Pat. No. 5,128,130 by Gilroy et al., U.S. Pat. No. 5,281,532 by Rammler et al., U.S. Pat. Nos. 4,695,455 and 4,861,595 by Barnes et al., U.S. Pat. No. 4,755,465 by Gray et al., and U.S. Pat. No. 5,169,760 by Wilcox.

[0075] III. Charge-shielded fusion proteins In some embodiments, the charge masked domain is located at the N-terminus of the fusion protein. In some embodiments, the charge masked domain is located at the C-terminus of the fusion protein. In some embodiments, the charge masked domain is located at the N-terminus of the biologically active domain. In some embodiments, the charge masked domain is located at the C-terminus of the biologically active domain. In some embodiments, the charge masked fusion protein comprises a peptide linker between the charge masked domain and the biologically active domain.

[0076] In some embodiments, the fusion proteins provided herein comprise a biologically active domain and a charge masking domain. In some embodiments, the charge masking domain prevents or reduces binding of the biologically active domain to an ion exchange chromatography resin. In some embodiments, the charge masking domain increases the hydrophobicity of the fusion protein. In some embodiments, the charge masking domain covers a charged region of the biologically active domain.

[0077] As used herein, a "bioactive domain" is a protein or peptide that has biological activity by itself or in association with another molecule, such as a protein, lipid, nucleic acid, or other monomer. For example, a "bioactive domain" includes a subunit of a multimeric protein complex.

[0078] In some embodiments, the charge shielding domain is uncharged. In some embodiments, the charge shielding domain has a pI of about 7, such as, for example, about 6.5 to about 7.5, about 6.6 to about 7.4, about 6.7 to about 7.3, about 6.8 to about 7.2, or about 6.9 to about 7.1. In some embodiments, the charge shielding domain has a pI of 5 to 9, 5 to 6, 5 to 7, 7 to 8, or 7 to 9. In some embodiments, the charge shielding domain comprises an uncharged amino acid. In some embodiments, the charge shielding domain comprises a polar amino acid. In some embodiments, the charge shielding domain comprises a nonpolar amino acid. In some embodiments, the charge shielding domain consists of proline, alanine, and serine. In some embodiments, the charge shielding domain consists of proline and alanine.

[0079] In some embodiments, the charge shielding domain has a molecular weight of 10 to 200 kDa, such as 10 to 100 kDa, 10 to 80 kDa, 10 to 60 kDa, or 10 to 40 kDa. In some embodiments, the charge shielding domain has a molecular weight of 10 to 20 kDa.

[0080] In some embodiments, the charge shielded fusion protein forms a multimeric protein, in some embodiments, the charge shielded protein forms a dimer, trimer, tetramer, hexamer, or octamer, in some embodiments, the charge shielded fusion protein forms a tetramer.

[0081] In some embodiments, the molecular weight of the charge-shielded multimeric (eg, tetrameric) protein is 50-500 kDa, 75-300 kDa, or 100-250 kDa.

[0082] In some embodiments, the molecular weight of the charge shielding domain is less than the molecular weight of the biologically active domain, hi some embodiments, the molecular weight of the charge shielding domain is less than 80%, less than 70%, less than 60%, less than 50%, less than 40%, less than 30%, or less than 20% of the molecular weight of the biologically active domain.

[0083] In some embodiments, the molecular weight of the charge shielding domain is greater than the molecular weight of the biologically active domain, ie, the molecular weight of the charge shielding domain is at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, or at least 200% of the molecular weight of the biologically active domain.

[0084] In some embodiments, the molecular weight of the charge shielding domain is about 25% to about 150% of the molecular weight of the biologically active domain. In some embodiments, the molecular weight of the charge shielding domain is about 50% to about 125% of the molecular weight of the biologically active domain. In some embodiments, the molecular weight of the charge shielding domain is about 50% to about 100% of the molecular weight of the biologically active domain.

[0085] In some embodiments, the total molecular weight of the charge shielded fusion protein is at least 50 kDa, at least 100 kDa, at least 120 kDa, or at least 150 kDa.

[0086] In some embodiments, the charge shielding domain adopts a random coil conformation. In some embodiments, the charge shielding domain adopts a random coil conformation in an aqueous environment (e.g., aqueous solution or aqueous buffer). The presence of a random coil conformation can be determined using methods known in the art, in particular by spectroscopic techniques such as circular dichroism (CD) spectroscopy. In some embodiments, the charge shielding domain has a disordered structure. In some embodiments, the charge shielding domain is unstructured.

[0087] In another embodiment, the charge shielding domain is characterized by having greater than 90% random coil formation, or greater than about 95%, or about 96%, or about 97%, or about 98%, or about 99% random coil formation as determined by the GOR algorithm. In some embodiments, the charge shielding domain has less than 20%, less than 15%, less than 10%, less than 5%, or less than 3% alpha helices. In some embodiments, the charge shielding domain has less than 20%, less than 15%, less than 10%, less than 5%, or less than 3% beta sheets. In some embodiments, the charge shielding domain has less than 2% alpha helices and less than 2% beta sheets as determined by the Chou-Fasman algorithm.

[0088] In another embodiment, the invention provides a fusion protein, wherein the charge shielding domain is characterized in that the sum of asparagine and glutamine residues is less than 10% of the entire amino acid sequence of the charge shielding domain, the sum of methionine and tryptophan residues is less than 2% of the entire amino acid sequence of the charge shielding domain, and the charge shielding domain sequence has less than 5% positively charged amino acid residues.

[0089] In another embodiment, the charge shielding domain is characterized in that at least about 80%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% of the charge shielding domain sequence consists of non-overlapping sequence motifs, each of the sequence motifs having from about 9 to about 14 amino acid residues, and wherein the sequence of any two consecutive amino acid residues does not occur more than twice in each of the sequence motifs consisting of 4 to 6 amino acids selected from glycine (G), alanine (A), serine (S), threonine (T), glutamic acid (E), and proline (P).

[0090] In some embodiments, the charge shielding domain increases the hydrodynamic radius of the fusion protein. The term "hydrodynamic radius" or "Stokes radius" is the effective radius (Rh in nm) of a molecule in solution, measured by assuming that it is a body moving through the solution and resisted by the viscosity of the solution. In an embodiment of the present invention, the hydrodynamic radius measurement of the fusion protein correlates with the "apparent molecular weight factor", which is a more intuitive measure. The "hydrodynamic radius" of a protein affects its rate of diffusion in aqueous solution and its ability to move in macromolecular gels. The hydrodynamic radius of a protein is determined by its molecular weight and its structure, including shape and compactness. Methods for determining hydrodynamic radius are well known in the art, such as by using size exclusion chromatography (SEC) as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Most proteins have a globular structure, which is the most compact three-dimensional structure in which a protein can have the smallest hydrodynamic radius. Some proteins adopt a random, open, unstructured, or "linear" conformation and, as a result, have a much larger hydrodynamic radius compared to typical globular proteins of similar molecular weight.

[0091] In some embodiments, the charge shielding domain can extend the hydrodynamic radius of the fusion protein beyond the glomerular pore size of about 3-5 nm (corresponding to an apparent molecular weight of about 70 kDA) (Caliceti. 2003. Pharmacokinetic and biodistribution properties of poly(ethylene glycol)-protein conjugates. Adv Drug Deliv Rev 55:1261-1277), resulting in reduced renal clearance of circulating proteins. The hydrodynamic radius of a protein is determined by its structure, including its molecular weight and shape or compactness. Methods for determining hydrodynamic radius are well known in the art, such as, for example, by the use of size exclusion chromatography (SEC) as described in U.S. Pat. Nos. 6,406,632 and 7,294,513. Thus, in certain embodiments, the fusion protein has a hydrodynamic radius of at least about 5 nm, or at least about 8 nm, or at least about 10 nm, or 12 nm, or at least about 15 nm. In the foregoing embodiments, the large hydrodynamic radius imparted by the charge shielding domain can result in reduced renal clearance of the resulting fusion protein, leading to a corresponding increase in terminal half-life, increased mean residence time, and / or reduced renal clearance rate.

[0092] In some embodiments, the charge masking domain does not affect the function of the biologically active domain, hi some embodiments, the biologically active domain retains at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of its activity when fused to a charge masking domain.

[0093] In some embodiments, the charge shielding domain increases the in vivo half-life of the fusion protein or multimers of charge shielded fusion protein subunits (i.e., dimers, trimers, tetramers, hexamers, or octamers). In some embodiments, the charge shielding domain increases the in vivo half-life by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 150%, or at least 200% compared to a biologically active protein without a charge shielding domain. In some embodiments, the charge shielding domain increases the in vivo half-life by at least 5-fold, at least 8-fold, at least 10-fold, at least 20-fold, or more than 30-fold. In some embodiments, the charge shielding domain increases the in vivo half-life by 5-50-fold, 5-40-fold, 5-30-fold, or 5-20-fold.

[0094] In some embodiments, the charge shielding domain is selected to confer an increase in the half-life of the fusion protein or multimer of the fusion protein (i.e., a dimer, trimer, tetramer, hexamer, or octamer) administered to an animal compared to a corresponding biologically active domain that is not linked to a charge shielding domain, and is administered at a dose that corresponds to an increase in half-life of at least about 2-fold longer, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold, or at least about 6-fold, or at least about 7-fold, or at least about 8-fold, or at least about 9-fold, or at least about 10-fold, or at least about 15-fold, or at least 20-fold, or at least 40-fold, or at least 80-fold, or at least 100-fold, or more, compared to a biologically active domain that is not linked to a charge shielding domain. In some embodiments, the invention provides fusion proteins that exhibit an increase in AUC of at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 150%, or at least about 200%, or at least about 300%, or at least about 500%, or at least about 1000%, or at least about 2000%, compared to a corresponding bioactive domain not linked to a charge shielding domain and administered to an animal at a comparable dose. Pharmacokinetic parameters of the fusion proteins can be determined by standard methods, including administration, taking blood samples at intervals, and assaying the protein using ELISA, HPLC, radioassay, or other methods known in the art or described herein, followed by standard calculations of the data to derive half-life and other PK parameters.

[0095] In addition, the fusion protein may have a half-life of at least about 5, 10, 12, 15, 24, 36, 48, 60, 72, 84, or 96 hours at a dose of about 25 μg / kg of protein.

[0096] In some embodiments, the charge shielding domain is a PAS domain. In some embodiments, the PAS domain consists of proline, alanine, and / or serine residues. In some embodiments, the PAS domain comprises 10-1000 amino acids. In some embodiments, the PAS domain comprises 100-1000 amino acids. In some embodiments, the PAS domain comprises 200-800 amino acids. In some embodiments, the PAS domain comprises 200-700 amino acids. In some embodiments, the PAS domain comprises 200-600 amino acids. In some embodiments, the PAS domain comprises 200-400 amino acids.

[0097] In some embodiments, the charge shielded fusion protein comprises a bioactive domain and a PAS domain. In some embodiments, "PASylated" or "PASylated" as used herein means that a bioactive domain is fused to a PAS domain.

[0098] In some embodiments, the PAS domain comprises 10 to 100 or more proline and alanine amino acid residues, a total of 15 to 60 proline and alanine amino acid residues, a total of 15 to 45 proline and alanine amino acid residues, for example, a total of 20 to about 40 proline and alanine amino acid residues, for example, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 proline and alanine amino acid residues. In a preferred embodiment, the amino acid sequence consists of about 20 proline and alanine amino acid residues. In another preferred embodiment, the amino acid sequence consists of about 40 proline and alanine amino acid residues.

[0099] A polypeptide consisting of only proline and alanine amino acid residues may have a length of about 200 to about 400 proline and alanine amino acid residues. In other words, the polypeptide may consist of about 200 to about 400 proline and alanine amino acid residues. In a preferred embodiment, the polypeptide consists of a total of about 200 (e.g., 201) proline and alanine amino acid residues (i.e., has a length of about 200 (e.g., 201) proline and alanine amino acid residues), or the polypeptide consists of a total of about 400 (e.g., 401) proline and alanine amino acid residues (i.e., has a length of about 400 (e.g., 401) proline and alanine amino acid residues). In some embodiments, the charge shielding domain consists of a random sequence of about 200 to about 400 proline and alanine residues.

[0100] The charge shielding domain may comprise multiple amino acid repeats, which consist of proline and alanine residues and are identical for no more than six consecutive amino acid residues. In particular, the polypeptide may comprise or consist of the amino acid sequence AAPAAPAPAAPAAPAPAAPA (SEQ ID NO:2) or a circularly modified form or multimer of the sequence as the whole or part of the sequence.

[0101] AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA ...

[0102] AAPAAPAPAAPAAPAPAAPAAAPAAPAPAAPAAPAPAAPAAAPAAPAPAA ...

[0103] In some embodiments, the bioactive domain is a hormone. In some embodiments, the bioactive domain is an enzyme. In some embodiments, the bioactive domain is an immunoglobulin. In some embodiments, the bioactive domain is a therapeutic peptide. In some embodiments, the bioactive domain is a therapeutic polypeptide.

[0104] In some embodiments, the bioactive domain is an antimicrobial peptide, including but not limited to agouti-related peptide, amylin, angiotensin, cecropin, bombesin, gastrin releasing peptide, including gastrin, lactoferrin, magainin, urodilatin, nuclear localization signal (NLS), collagen peptides, survivin, amyloid peptides, including f-amyloid, natriuretic peptides, peptide YY, neuropeptide Y, neuroregenerative peptides and neuropeptides, including but not limited to dynorphins, endomorphins, endothelins, enkaphalins, exendins, fibronectin, neuropeptide W and neuropeptide S, peptide T, melanocortin, amyloid precursor protein, sheet breaker peptides, CART13 WO2008 / 030968 PCT / US2007 / 077767 peptides, amyloid inhibitory peptides, prion inhibitory peptides, chlorotoxin, corticotropin releasing factor, oxytocin, vasopressin, cholecystokinin, secretin, thymosin, epidermal growth factor (EGF), vascular endothelial growth factor (VEGF), platelet derived growth factor (PDGF), insulin-like growth factor (IGF), fibroblast growth factor (aFGF, bFGF), pancreastatin, melanocyte stimulating hormone, osteocalcin, bradykinin, adrenomedullin, perinelin, metastatin, aprotinin, galanin including galanin-like peptides, leptin, a-defensins and f defensins, including but not limited to defensins, salusins, and various venoms, including but not limited to conotoxins, decorsin, kurtoxins, anemone venom, and tarantula venom; natriuretic peptides, including brain natriuretic peptide (B-type natriuretic peptide, or BNP), atrial natriuretic peptide, and vasonathrin; neurokinin A, neurokinin B; neuromedin; neurotensin; orexin, pancreatic polypeptide, pituitary adenylate cyclase activating peptide (PACAP), prolactin-releasing peptide, proteolipid protein (PLP), somatostatin, and TNF-a;Ghrelin, Protein C (Xigris), SS1(dsFv)-PE38 and pseudomonas exotoxin protein, antithrombin III and clotting factors including clotting factors VIIA, VIII and IX, streptokinase, tissue plasminogen activator, urokinase, beta-glucocerebrosidase and alpha-D-galactosidase, alpha-L-iduronidase, alpha-1,4-glucosidase, arylsulfatase B, iduronate-2-sulfatase, deoxyribonuclease I, human activator protein, follicle-stimulating hormone, chorionic gonadotropin, luteinizing hormone, somatropin, bone morphogenetic protein, nesiritide, adjuvant thyroid hormone, erythropoietin, keratinocyte growth factor, human granulocyte colony stimulating factor (G-CSF), human granulocyte macrophase colony stimulating factor (GM-CSF), alpha interferon, beta interferon, gamma interferon, interleukins including IL-1, IL-iRa, IL-2, IL-11-4, IL-5, IL-6, IL-10, IL11, IL-12, immunoglobulins including glycoprotein IIB / IIIA, hepatitis B, gamma globulins, venolglobulins, hirudin, aprotinin, antithrombin III, alpha-i-proteinase inhibitors, filgrastim, and etanercept, or variants, fragments, or derivatives thereof;

[0105] In another embodiment, the bioactive domain is an antibody or antigen, which is relevant for immunotherapy or other therapeutic intervention.

[0106] In some embodiments, the bioactive domain is selected from the group consisting of insulin A peptide, T20 peptide, interferon alpha 2B peptide, tobacco etch virus protease, small heterodimer partner orphan receptor, androgen receptor ligand binding domain, glucocorticoid receptor ligand binding domain, estrogen receptor ligand binding domain, G protein alpha Q, 1-deoxy-D-xylulose 5-phosphate reductoisomerase peptide, G protein alpha S, angiostatin (Ki-3), blue fluorescent protein (BFP), calmodulin (CalM), chloramphenicol acetyltransferase (CAT), green fluorescent protein (GFP), interleukin I receptor antagonist (IL-iRa), luciferase, tissue transglutaminase (tTg), morphine regulatory neuropeptide 14 WO2008 / 030968 PCT / US2007 / 077767 (MMN), neuropeptide Y (NPY), orexin-B, leptin, ACTH, calcitonin, adrenomedullin (AM), parathyroid hormone (PTH), defensins, and growth hormone.

[0107] In some embodiments, the bioactive domain has a molecular weight of less than 200 kDa. In some embodiments, the bioactive domain has a molecular weight of less than about 150 kDa. In some embodiments, the bioactive domain has a molecular weight of less than about 100 kDa. In some embodiments, the bioactive domain has a molecular weight of less than about 70 kDa, which is the threshold for renal filtration. In some embodiments, the bioactive domain has a molecular weight of less than about 50 kDa.

[0108] In some embodiments, the bioactive domain has a molecular weight of about 20 to about 100 kDa. In some embodiments, the bioactive domain has a molecular weight of about 20 to about 70 kDa. In some embodiments, the bioactive domain has a molecular weight of about 30 to about 40 kDa.

[0109] In some embodiments, the biologically active domain can form a multimer. In some embodiments, the biologically active domain can form a dimer, trimer, tetramer, hexamer, or octamer. In some embodiments, the molecular weight of the multimeric biologically active domain is about 20 kDa to about 300 kDa, about 50 kDa to about 200 kDa, or about 100 kDa to about 200 kDa.

[0110] In some embodiments, the bioactive domain has a net charge in neutral solution. In some embodiments, the bioactive domain has a pI other than 7.0. In some embodiments, the bioactive domain has a pI of about 3.0 to about 6.0, about 4.0 to about 6.0, or about 5.0 to about 6.0. In some embodiments, the bioactive domain has a pI of about 8.0 to about 10.0, or about 8.0 to about 9.0.

[0111] In some embodiments, the bioactive domain is an enzyme. In some embodiments, the bioactive domain is an asparaginase subunit. Recombinant type II asparaginase, crisantaspase, from Erwinia chrysanthemi (blackleg disease) is also known as Erwinase® and Erwinaze®. Recombinant asparaginase from E. coli is known by the names Colaspase®, Elspar®, Kidrolase®, Leunase®, and Spectrila®. Pegaspargase® is the name for the pegylated form of E. coli asparaginase. Crisantaspase is administered via intravenous, intramuscular, or subcutaneous injection to patients with acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin's lymphoma.

[0112] In some embodiments, the asparaginase is Erwinia chrysanthemi L-asparaginase type II (crisantaspase). In some embodiments, the asparaginase comprises the amino acid sequence ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY. (SEQ ID NO: 1)

[0113] In some embodiments, the asparagine is a recombinant E. coli asparaginase. E. coli produces two asparaginases: L-asparaginase type I and L-asparaginase type II. L-asparaginase type I has a low affinity for asparagine and is localized in the cytoplasm. L-asparaginase type II is a tetrameric periplasmic enzyme with a high affinity for asparagine that is produced with a cleavable secretory leader sequence. U.S. Patent Application Publication No. 2016 / 0060613, "Pegylated L-asparaginase," incorporated by reference in its entirety, describes the common structural features of known L-asparaginases from bacterial sources. According to US2016 / 0060613, all are homotetramers with four active sites between the N- and C-terminal domains of two adjacent monomers, all have a high degree of similarity in their tertiary and quaternary structures, and the sequence of the catalytic site of L-asparaginase is highly conserved between Erwinia chrysanthemi, Erwinia carotovora (soft rot bacteria), and E. coli L-asparaginase II.

[0114] In embodiments, the E. coli A-1-3 L-asparaginase type II comprises the amino acid sequence: LPNITILATGGTIAGGGDSATKSNYTAGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDDVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVDAGYDGIVSAGVGNGNLYKTVFDTLATAAKNGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY (SEQ ID NO:5).

[0115] In some embodiments, asparaginase is produced using the methods of the present invention. This asparaginase is described, for example, in U.S. Patent No. 7,807,436, "Recombinant host for producing L-asparaginase II," the sequence of which is set forth as SEQ ID NO: 5, which is incorporated herein by reference in its entirety. E. coli A-1-3 L-asparaginase type II is also described by Nakamura, N., et al., 1972, "On the Productivity and Properties of L-Asparaginase from Escherichia coli A-1-3," Agricultural and Biological Chemistry, 36:12, 2251-2253, which is incorporated herein by reference. E. coli A-1-3 is derived from the E. coli HAP strain, which produces high levels of asparagine, and is described in Roberts, J., et al., 1968, "New Procedures for Purification of L-Asparaginase with High Yield from Escherichia coli," Journal of Bacteriology, 95:6, 2117-2123, which is incorporated herein by reference.

[0116] In an embodiment, the L-asparaginase type II protein produced using the methods of the present invention is the E. coli K-12 L-asparaginase type II enzyme, as described in Jennings et al., 1990, J. Bacteriol. 172:1491-1498 (GenBank No. M34277), both of which are incorporated herein by reference (MEFFKKTALAALVMGFSGAALALPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKA The amino acid sequence shown as RVLLQLALTQTKDPQQIQQIFNQY (SEQ ID NO: 6) or LPNITILATGGTIAGGGDSATKSNYTVGKVGVENLVNAVPQLKDIANVKGEQVVNIGSQDMNDNVWLTLAKKINTDCDKTDGFVITHGTDTMEETAYFLDLTVKCDKPVVMVGAMRPSTSMSADGPFNLYNAVVTAADKASANRGVLVVMNDTVLDGRDVTKTNTTDVATFKSVNYGPLGYIHNGKIDYQRTPARKHTSDTPFDVSKLNELPKVGIVYNYANASDLPAKALVDAGYDGIVSAGVGNGNLYKSVFDTLATAAKTGTAVVRSSRVPTGATTQDAEVDDAKYGFVASGTLNPQKARVLLQLALTQTKDPQQIQQIFNQY (SEQ ID NO: 7) (without the leader sequence)

[0117] U.S. Patent No. 7,807,436 reports that compared to the L-asparaginase type II enzyme from Merck & Co., Inc. (Elspar®) and the L-asparaginase type II enzyme from Kyowa Hakko Kogyo Co., Ltd., the E. coli K12 enzyme subunit has Val27 instead of Ala27, Asn64 instead of Asp64, Ser252 instead of Thr252, and Thr263 instead of Asn263.

[0118] In an embodiment, the L-asparaginase type II produced using the methods of the present invention has an amino acid sequence as set forth by Maita, T., et al, December 1974, "Amino acid sequence of L-asparaginase from Escherichia coli," J. Biochem. 76(6):1351-4, which is incorporated herein by reference.

[0119] Recombinant type II asparaginase from E. coli is also known by the names Colaspase®, Elspar®, Kidrolase®, Leunase®, and Spectrila®. Pegaspargase® is the name for the pegylated form of E. coli asparaginase. Asparaginase is administered via intravenous, intramuscular, or subcutaneous injection to patients with acute lymphoblastic leukemia, acute myeloid leukemia, and non-Hodgkin's lymphoma.

[0120] In some embodiments, the fusion protein comprises an asparaginase subunit having at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:7. In some embodiments, the fusion protein comprises an asparaginase subunit comprising SEQ ID NO:7 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid substitutions. In some embodiments, the amino acid substitutions are conservative substitutions. In some embodiments, the fusion protein comprises an asparaginase subunit comprising SEQ ID NO:7 with 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid insertions or deletions.

[0121] Substitutions include conservative amino acid substitutions. A "conservative amino acid substitution" is one in which an amino acid residue is replaced with an amino acid residue having a similar side chain or physicochemical characteristics (e.g., electrostatic, hydrogen bonding, isosteric, hydrophobic properties). The amino acid can be naturally occurring or non-naturally occurring. Families of amino acid residues with similar side chains are known in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, methionine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Substitutions can also include non-conservative changes.

[0122] Erwinia chrysanthemi NCPPB1066 (Genbank Accession No. CAA32884, described by, e.g., Minton, et al., 1986, "Nucleotide sequence of the Erwinia chrysanthemi NCPPB 1066 L-asparaginase gene," Gene 46(1), 25-35, each of which is incorporated by reference in its entirety), with or without a signal peptide and / or leader sequence.

[0123] In some embodiments, the fusion protein comprises an asparaginase from Dickeya chrysanthemi. In some embodiments, the asparaginase comprises the amino acid sequence ADKLPNIVILATGGTIAGSAATGTQTTGYKAGALGVDTLINAVPEVKKLANVKGEQFSNMASENMTGDVVLKLSQRVNELLARDDVDGVVITHGTDTVEESAYFLHLTVKSDKPVVFVAAMRPATAISADGPMNLLEAVRVAGDKQSRGRGVMVVLNDRIGSARYITKTNASTLDTFKANEEGYLGVIIGNRIYYQNRIDKLHTTRSVFDVRGLTSLPKVDILYGYQDDPEYLYDAAIQHGVKGIVYAGMGAGSVSVRGIAGMRKAMEKGVVVIRSTRTGNGIVPPDEELPGLVSDSLNPAHARILLMLALTRTSDPKVIQEYFHTY (SEQ ID NO: 8).

[0124] In some embodiments, the fusion protein comprises the amino acid sequence (SEQ ID NO:9).

[0125] In some embodiments, the fusion protein comprises the amino acid sequence (SEQ ID NO:10).

[0126] IV. Production of Charge-Shielded Recombinant Erwinia Fusion Proteins In some embodiments, the method includes expressing and purifying a charge shielded asparaginase fusion protein. In some embodiments, the method includes expressing a type II asparaginase fusion protein. In some embodiments, the asparaginase is Erwinia chrysanthemi L-asparaginase type II (crisantaspase). In some embodiments, the asparaginase fusion protein is expressed in a prokaryotic host cell. In some embodiments, the asparaginase fusion protein is expressed in a Pseudomonas fluorescens host cell. In some embodiments, the Pseudomonadales host cell is deficient in expression of one or more native asparaginases. In some embodiments, the native asparaginase deficient in expression is a type I asparaginase. In some embodiments, the native asparaginase deficient in expression is a type II asparaginase. In some embodiments, the Pseudomonadales host cell is deficient in expression of one or more proteases. In some embodiments, the Pseudomonadales host cell overexpresses one or more folding regulators. In some embodiments, the Pseudomonadales host cell is deficient in expression of one or more native asparaginases, is deficient in expression of one or more proteases, and / or overexpresses one or more folding regulators. US10787671 provides methods for producing recombinant Erwinia asparaginase.

[0127] In its native host, Erwinia chrysanthemi, crisantaspase is produced in the periplasm. The present invention provides methods that allow for the production of high levels of soluble and / or active crisantaspase in the cytoplasm of host cells. In embodiments, the methods provided herein result in high levels of soluble and / or active crisantaspase in the cytoplasm of Pseudomonadales, Pseudomonad, Pseudomonas, or Pseudomonas fluorescens host cells.

[0128] In some embodiments, the charge masked fusion protein is purified from the periplasmic release. In some embodiments, the nucleic acid encoding the charge masked fusion protein comprises a periplasmic secretion leader sequence.

[0129] In some embodiments, osmotic shock is used to generate the periplasmic release. In some embodiments, the cells are incubated with lysozyme to generate the periplasmic release. In some embodiments, the cells are sonicated to generate the periplasmic release. In some embodiments, the cells are incubated with lysozyme and sonicated to generate the periplasmic release.

[0130] In some embodiments, chemicals such as chloroform (Ames et al. (1984) J. Bacteriol., 160:1181-1183), guanidine-HCl, and Triton X-100 (Naglak and Wang (1990) Processes including Enzyme Microb. Technol., 12:603-611) have been used to release charge-shielded fusion proteins from the periplasm. However, these chemicals are not inert and may adversely affect many recombinant protein products or subsequent purification procedures. Glycine treatment of E. coli cells, which results in increased permeability of the outer membrane, has also been reported to release periplasmic contents (Ariga et al. (1989) J. Ferm. Bioeng., 68:243-246). The most widely used methods of recombinant protein periplasmic release are osmotic shock (Nosal and Heppel (1966) J. Biol. Chem., 241:3055-3062; Neu and Heppel (1965) J. Biol. Chem., 240:3685-3692), hen egg white (HEW) lysozyme / ethylenediaminetetraacetic acid (EDTA) treatment (Neu and Heppel (1964) J. Biol. Chem., 239:3893-3900; Witholt et al. (1976) Biochim. Biophys. Acta, 443:534-544; Pierce et al. (1995) I Cheme Research. Event, 2:995-997), and combined HEW lysozyme / osmotic shock treatment (French et al. (1996) Enzyme and Microb. Tech., 19:332-338. The French method involves resuspension of cells in fractionation buffer followed by recovery of the periplasmic fraction, followed immediately by lysozyme treatment followed by osmotic shock.

[0131] Typically, these procedures involve an initial disruption in an osmotically stabilizing medium, followed by selective release in a non-stabilizing medium. The composition of these media (pH, protective agents) and the disruption method used (chloroform, HEW lysozyme, EDTA, sonication) depend on the particular procedure reported. HEW using a zwitterionic detergent instead of EDTA A change in lysozyme / EDTA treatment is described in Statel et al. (1994) Veterinary Microbiol., 38:307-314. For a general review of the use of endolytic enzyme systems to disrupt E. coli, see Dabora and Cooney (1990) in Advances in Biochemical Engineering / Biotechnology, Vol. 43, A. Fiechter, ed. (Springer-Verlag: Berlin), pp. See 11-30.

[0132] In some embodiments, the charge shielded asparaginase fusion protein is expressed in an expression construct, such as a plasmid, without a secretion signal. An inducible promoter sequence is used to regulate the expression of crisantaspase according to the methods herein. In embodiments, inducible promoters useful in the methods herein include those of the family derived from the lac promoter (i.e., the lacZ promoter), in particular the tac and trc promoters described in U.S. Patent No. 4,551,433 by DeBoer, as well as the Ptac16, Ptac17, PtacII, PlacUV5, and T7lac promoters. In one embodiment, the promoter is not derived from the host cell organism. In certain embodiments, the promoter is derived from the E. coli organism. In some embodiments, the lac promoter is used to regulate the expression of crisantaspase from a plasmid. In the case of lac promoter derivatives or family members, such as the tac promoter, the inducer is IPTG (isopropyl-β-D-1-thiogalactopyranoside, also called "isopropylthiogalactoside"). In certain embodiments, IPTG is added to the culture to induce expression of crisantaspase from the lac promoter in the Pseudomonas host cells.

[0133] Expression constructs useful for practicing the methods herein include, in addition to the protein coding sequence, the following regulatory elements operably linked thereto: a promoter, a ribosome binding site (RBS), a transcription terminator, and translation start and stop signals.

[0134] Pseudomonas and closely related bacteria are part of the group generally defined as "Gram(-) Proteobacteria Subgroup 1" or "Gram-negative aerobic bacilli and cocci" (Bergey's Manual of Systematics of Archaea and Bacteria (online publication, 2015)). Pseudomonas host strains are described in the literature, such as U.S. Patent Application Publication No. 2006 / 0040352, cited above.

[0135] "Gram-negative Proteobacteria subgroup 1" also includes Proteobacteria that are classified under this heading according to the criteria used in classification. The heading also includes groups that were previously classified in this section but are no longer classified, such as the genera Acidovorax, Brevundimonas, Burkholderia, Hydrogenophaga, Oceanimonas, Ralstonia, and Stenotrophomonas, the genus Sphingomonas (and Blastomonas derived therefrom) created by reclassifying organisms within the genus Xanthomonas, and the genus Acidomonas created by reclassifying organisms within the genus Acetobacter as defined in Bergey's Manual of Systematics of Archaea and Bacteria (online publication, 2015). In addition, hosts include cells from the genus Pseudomonas, Pseudomonas enalia (ATCC14393), Pseudomonas nigrifaciensi (ATCC19375), and Pseudomonas putrefaciens (ATCC8071), which have been reclassified as Alteromonas haloplanktis, Alteromonas nigrifaciens, and Alteromonas putrefaciens, respectively.Similarly, for example, Pseudomonas acidovorans (ATCC 15668) and Pseudomonas testosteroni (ATCC 11996) have since been reclassified as Comamonas acidovorans and Comamonas testosteroni, respectively, and Pseudomonas nigrifaciens (ATCC 19375) and Pseudomonas piscicida (ATCC 15057) have been reclassified as Pseudoalteromonas nigrifaciens and Pseudoalteromonas piscicida, respectively. "Gram-negative Proteobacteria Subgroup 1" also includes Proteobacteria classified as belonging to any of the families: Pseudomonadaceae, Azotobacteraceae (now often referred to by the synonym "nitrogen fixing group" of Pseudomonadaceae), Rhizobiaceae, and Methylomonadaceae (now often referred to by the synonym "Methylococcaceae").As a result, in addition to these genera, in another method described herein, further Proteobacterial genera falling within "Gram-negative Proteobacteria subgroup 1" include: 1) Azotobacter group bacteria of the genus Azorhizophilus; 2) Pseudomonadaceae family bacteria of the genera Cellvibrio, Oligella, and Teredinibacter; 3) bacteria of the genera Chelatobacter, Ensifer, Liberibacter (also known as "Candidatus"); and 3) bacteria of the genera Methylobacter, Methylocaldum, Methylomicrobium, Methylosarcina, and Methylosphaera, which belong to the family Methylococcaceae.

[0136] The host cell, in some cases, is selected from "Gram-negative Proteobacteria subgroup 16." "Gram-negative Proteobacteria Subgroup 16" includes the following Pseudomonas species (with ATCC or other deposit number of exemplary strains shown in parentheses): Pseudomonas abietaniphila (ATCC 700689), Pseudomonas aeruginosa (ATCC 10145), Pseudomonas alcaligenes (ATCC 14909), Pseudomonas anguilliseptica (ATCC 33660), Pseudomonas citronellolis (ATCC 13674), Pseudomonas flavescens (ATCC 51555), Pseudomonas mendocina (ATCC25411), Pseudomonas nitroreducens (ATCC33634), Pseudomonas oleovorans (ATCC8062), Pseudomonas pseudoakaligenes (ATCC17440), Pseudomonas resinovorans (ATCC14235), Pseudomonas straminea (ATCC33636), Pseudomonas agarici (ATCC25941), Pseudomonas alcaliphila, Pseudomonas alginovora, Pseudomonas andersonii, Pseudomonas asplenii (ATCC23835), PseudomonasPseudomonas azelaica (ATCC27162), Pseudomonas beyerinckii (ATCC19372), Pseudomonas borealis, Pseudomonas boreopolis (ATCC33662), Pseudomonas brassicacearum, Pseudomonas butanovora (ATCC43655), Pseudomonas cellulosa (ATCC55703), Pseudomonas aurantiaca (ATCC33663), Pseudomonas chlororaphis (ATCC9446, ATCC 13985, ATCC 17418, ATCC 17461), Pseudomonas fragi (ATCC4973), Pseudomonas lundensis (ATCC49968), Pseudomonas taetrolens (ATCC4683), Pseudomonas cissicola (ATCC33616), Pseudomonas coronafaciens, Pseudomonas diterpeniphila, Pseudomonas Microbulbifer elongata (ATCC10144), Pseudomonas flectens (ATCC12775), Pseudomonas azotoformans, Pseudomonas brenneri, Pseudomonas cedrella, PseudomonasPseudomonas corrugata (ATCC29736), Pseudomonas extremorientalis, Pseudomonas fluorescens (ATCC35858), Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii (ATCC700871), Pseudomonas marginalis (ATCC10844), Pseudomonas migulae, Pseudomonas mucidolens (ATCC4685), Pseudomonas orientalis, Pseudomonas rhodesiae, Pseudomonas synxantha (ATCC9890), Pseudomonas tolaasii (ATCC33618), Pseudomonas veronii (ATCC700474), Pseudomonas frederiksbergensis, Pseudomonas geniculata (ATCC19374), Pseudomonas gingeri, Pseudomonas graminis, Pseudomonas grimontii, Pseudomonas halodenitrificans, Pseudomonas halophila, Pseudomonas hibiscicola (ATCC19867), PseudomonasPseudomonas huttiensis (ATCC14670), Pseudomonas hydrogenovora, Pseudomonas jessenii (ATCC700870), Pseudomonas kilonensis, Pseudomonas lanceolata (ATCC14669), Pseudomonas lini, Pseudomonas marginata (ATCC25417), Pseudomonas mephitica (ATCC33665), Pseudomonas denitrificans (ATCC19244), Pseudomonas Pseudomonas pertucinogena (ATCC190), Pseudomonas pictorum (ATCC23328), Pseudomonas psychrophila, Pseudomonas filva (ATCC31418), Pseudomonas monteilii (ATCC700476), Pseudomonas mosselii, Pseudomonas oryzihabitans (ATCC43272), Pseudomonas plecoglossicida (ATCC700383), Pseudomonas Pseudomonas putida (ATCC12633), Pseudomonas reactans, Pseudomonas spinosa (ATCC14606), Pseudomonas balearica, PseudomonasPseudomonas luteola (ATCC43273), Pseudomonas stutzeri (ATCC17588), Pseudomonas amygdali (ATCC33614), Pseudomonas avellanae (ATCC700331), Pseudomonas caricapapayae (ATCC33615), Pseudomonas cichorii (ATCC10857), Pseudomonas ficuserectae (ATCC35104), Pseudomonas fuscovaginae, Pseudomonas meliae (ATCC33050), Pseudomonas syringae (ATCC19310), Pseudomonas viridiflava (ATCC13223), Pseudomonas thermocarboxydovorans (ATCC35961), Pseudomonas thermotolerans, Pseudomonas thivervalensis, Pseudomonas vancouverensis (ATCC700688), Pseudomonas wisconsinensis, and Pseudomonas Pseudomonas xiamenensis is defined as a group of proteobacteria. In one embodiment, the host cell for expression of crisantaspase is Pseudomonas fluorescens.

[0137] The host cell, in some cases, is selected from "Gram-negative Proteobacteria Subgroup 17." "Gram-negative Proteobacteria Subgroup 17" includes, for example, those belonging to the following Pseudomonas species: Pseudomonas azotoformans, Pseudomonas brenneri, Pseudomonas cedrella, Pseudomonas cedrina, Pseudomonas corrugata, Pseudomonas extremorientalis, Pseudomonas fluorescens, Pseudomonas gessardii, Pseudomonas libanensis, Pseudomonas mandelii, Pseudomonas marginalis, Pseudomonas migulae, Pseudomonas mucidolens, Pseudomonas orientalis, Pseudomonas rhodesiae, Pseudomonas synxantha, Pseudomonas tolaasii, and Pseudomonas veronii. The fluorescent pseudomonads are defined as a group of Proteobacteria known in the art as "fluorescent pseudomonads."

[0138] In an embodiment, a host strain useful for expressing a charge-shielded crisantaspase fusion protein in the method of the invention is a Pseudomonas host strain, e.g., P. fluorescens, with a protease deficiency or inactivation (e.g., resulting from a deletion, partial deletion, or knockout) and / or overexpressing a folding regulator, e.g., from a plasmid or bacterial chromosome. In an embodiment, the host strain expresses the auxotrophic markers pyrF and proC, has a protease deficiency, and / or overexpresses a folding regulator. In an embodiment, the host strain expresses any other suitable selection marker known in the art. In any of the above embodiments, asparaginase, e.g., native type I and / or type II asparaginase, is inactivated in the host strain. In one embodiment, the method herein includes expression of a charge-shielded recombinant crisantaspase fusion protein from a construct that is optimized for codon usage in the strain of interest. In an embodiment, the strain is a Pseudomonas host cell, e.g., Pseudomonas fluorescens. Methods for codon optimization to improve expression in bacterial hosts are known in the art and described in the literature.

[0139] Growth conditions useful for the methods herein often include a temperature of about 4° C. to about 42° C., and a pH of about 5.7 to about 8.8. When an expression construct having a lacZ promoter or a derivative thereof is used, expression is often induced by adding IPTG to the culture to a final concentration of about 0.01 mM to about 1.0 mM.

[0140] II. Charge-shielded proteins As described elsewhere herein, an inducible promoter is often used in an expression construct to control expression of the charge-shielded recombinant crisantaspase fusion protein, for example, the lac promoter. In the case of a lac promoter derivative or family member, for example the tac promoter, the effector compound is an inducer, such as a gratuitous inducer such as IPTG (isopropyl-β-D-1-thiogalactopyranoside, also called "isopropylthiogalactoside"). In an embodiment, a lac promoter derivative is used and expression of the charge-shielded crisantaspase fusion protein is induced by the addition of IPTG to a final concentration of about 0.01 mM to about 1.0 mM when the cell density reaches a level specified by an OD575 of about 25 to about 160.

[0141] After adding the inducing agent, the culture is often grown for a period of time, for example, about 24 hours, during which the charge-shielded recombinant crisantaspase fusion protein is expressed. After adding the inducing agent, the culture is often grown for 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, about 20 hours, about 21 hours, about 22 hours, about 23 hours, about 24 hours, about 36 hours, or about 48 hours. After adding the inducing agent to the culture, the culture is grown for about 1-48 hours, about 1-24 hours, about 10-24 hours, about 15-24 hours, or about 20-24 hours. Cell cultures are often concentrated by centrifugation and the culture pellet is resuspended in an appropriate buffer or solution for the subsequent lysis procedure.

[0142] In embodiments, cells are disrupted using equipment for high pressure mechanical cell disruption (commercially available, e.g., Microfluidics microfluidic device, stationary cell disrupter, Niro-Soavi homogenizer, or APV-Gaulin homogenizer). Cells expressing charge-shielded crisantaspase fusion proteins are often disrupted, e.g., using sonication. Any suitable method known in the art for lysing cells is used to release the soluble fraction. For example, in embodiments, chemical and / or enzymatic cell lysis reagents, such as cell wall lytic enzymes and EDTA, are often used. The use of frozen or previously stored cultures is also contemplated in the methods herein. Cultures are sometimes OD normalized prior to lysis. For example, cells are often normalized to an OD600 of about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0143] Centrifugation is performed using appropriate equipment and methods. Centrifugation of cell culture or lysate or periplasmic release for the purpose of separating the soluble fraction from the insoluble fraction is well known in the art. For example, lysed cells are sometimes centrifuged at 20,800×g for 20 minutes (at 4° C.) and the supernatant is removed using manual or automated liquid handling. The pellet (insoluble) fraction is resuspended in a buffered solution, e.g., phosphate buffered saline (PBS), pH 7.4. Resuspension is often performed using equipment such as an impeller connected to an overhead mixer, a magnetic stir bar, or a rocking shaker.

[0144] In one embodiment, fermentation is used in the method for producing a charge-shielded recombinant crisantaspase fusion protein. The expression system according to the present disclosure is cultured in any fermentation format. For example, batch, fed-batch, semi-continuous, and continuous fermentation modes may be used herein. In an embodiment, the fermentation medium may be selected from among a rich medium, a minimal medium, and a mineral salts medium. In other embodiments, either a minimal medium or a mineral salts medium is selected. In a particular embodiment, a mineral salts medium is selected.

[0145] Fermentation can be carried out at any scale. The expression system according to the present disclosure is useful for recombinant protein expression at any scale. Thus, for example, fermentation volumes at the microliter scale, milliliter scale, centiliter scale, and deciliter scale can be used, with 1 liter scale and larger fermentation volumes often being used.

[0146] In embodiments, the methods herein are used to obtain a yield of soluble, charge-shielded, recombinant crisantaspase fusion protein, e.g., monomeric or tetrameric, of about 1% to about 70% of the total cell protein. In certain embodiments, the yield of soluble, charge-shielded, recombinant crisantaspase fusion protein is about 1% of the total cell protein, about 2% of the total cell protein, about 3% of the total cell protein, about 4% of the total cell protein, about 5% of the total cell protein, about 8% of the total cell protein, about 10% of the total cell protein, about 15% of the total cell protein, about 20% of the total cell protein, about 25% of the total cell protein, about 30% of the total cell protein, about 35% of the total cell protein, about 40% of the total cell protein, about 41% of the total cell protein, about 42% of the total cell protein, about 43% of the total cell protein, about 44% of the total cell protein, about 45% of the total cell protein, about 46% of the total cell protein, about 47% of the total cell protein, about 48% of the total cell protein, about 49% of the total cell protein, about 50% of the total cell protein, about 51% of the total cell protein, about 52% of the total cell protein, about 53% of the total cell protein, about 54% of the total cell protein, about 55% of the total cell protein, about 56% of the total cell protein, about 57% of the total cell protein, about 58% of the total cell protein, about 59% of the total cell protein, about 60% of the total cell protein, about 61% of the total cell protein, about 62% of the total cell protein, about 63% of the total cell protein, about 64% of the total cell protein, about 65% of the total cell protein, about 66% of the total cell protein % whole cell protein, about 47% whole cell protein, about 48% whole cell protein, about 49% whole cell protein, about 50% whole cell protein, about 51% whole cell protein, about 52% whole cell protein, about 53% whole cell protein, about 54% whole cell protein, about 55% whole cell protein, about 56% whole cell protein, about 57% whole cell protein, about 58% whole cell protein, about 59% whole cell protein, about 60% whole cell protein, about 65% whole cell protein, about 70% whole cell protein, about 75% whole cell protein, about 80% whole cell protein, about 85% whole cell protein, or about 90% whole cell protein.

[0147] In some embodiments, the yield of soluble, charge-shielded recombinant crisantaspase fusion protein is about 1% to about 70% whole cell protein, about 1% to about 50% whole cell protein, about 1% to about 20% whole cell protein, about 1% to about 10% whole cell protein, about 1% to about 5% whole cell protein, about 1% to about 3% whole cell protein, about 20% to about 55% whole cell protein, about 20% to about 60% whole cell protein, about 20% to about 65% whole cell protein, about 20% to about 70% whole cell protein, about 20% to about 75% whole cell protein, about 20% to about 80% whole cell protein, about 20% to about 85% whole cell protein, about 20% to about 90% whole cell protein, about 25% to about 90% whole cell protein, about 30% to about 90% whole cell protein, about 35% to about 35 ... % whole cell protein, about 35% to about 90% whole cell protein, about 40% to about 90% whole cell protein, about 45% to about 90% whole cell protein, about 50% to about 90% whole cell protein, about 55% to about 90% whole cell protein, about 60% to about 90% whole cell protein, about 65% to about 90% whole cell protein, about 70% to about 90% whole cell protein, about 75% to about 90% whole cell protein, about 80% to about 90% whole cell protein, about 85% to about 90% whole cell protein, about 1% to about 5% whole cell protein, about 2% to about 5% whole cell protein, about 5% to about 10% whole cell protein, about 20% to about 35% whole cell protein, about 20% to about 30% whole cell protein, or about 20% to about 25% whole cell protein. In some embodiments, the yield of soluble, charge-shielded recombinant crisantaspase fusion protein is about 20% to about 40% of the total cellular protein.

[0148] In embodiments, the methods herein are used to obtain a yield of soluble, charge-shielded, recombinant crisantaspase fusion protein, e.g., monomer or tetramer, of about 1 gram per liter to about 50 grams per liter. In certain embodiments, the yield of soluble, charge-shielded, recombinant crisantaspase fusion protein is about 0.25 grams per liter, about 0.5 grams per liter, about 1 gram per liter, about 2 grams per liter, about 3 grams per liter, about 4 grams per liter, about 5 grams per liter, about 6 grams per liter, about 7 grams per liter, about 8 grams per liter, about 9 grams per liter, about 10 grams per liter, about 11 grams per liter, about 12 grams per liter, about 13 grams per liter, about 14 grams per liter, about 15 grams per liter, about 16 grams per liter, about 17 grams per liter, about 18 grams per liter, about 19 grams per liter, about 20 grams per liter, about 21 grams per liter, about 22 grams per liter, about 23 grams per liter, about 24 grams per liter, about 25 grams per liter, about 26 grams per liter, about 27 grams per liter, about 28 grams per liter, about 29 grams per liter, about 30 grams per liter, about 31 grams per liter, about 32 grams per liter, about 33 grams per liter, about 34 grams per liter, about 35 grams per liter, about 36 grams per liter, about 37 grams per liter, about 38 grams per liter, about 39 grams per liter, about 40 grams per liter, about 41 grams per liter, about 42 grams per liter, about 43 grams per liter, about 44 grams per liter, about 45 grams per liter, about 4 about 15 grams per liter, about 16 grams per liter, about 17 grams per liter, about 18 grams per liter, about 19 grams per liter, about 20 grams per liter, about 21 grams per liter, about 22 grams per liter, about 23 grams per liter, about 24 grams per liter, about 25 grams per liter, about 26 grams per liter, about 27 grams per liter, about 28 grams per liter, about 30 grams per liter, about 35 grams per liter, about 40 grams per liter, about 45 grams per liter, and about 50 grams per liter.

[0149] In some embodiments, the yield of soluble, charge shielded, recombinant crisantaspase fusion protein is about 0.1 to about 6 grams per liter, about 0.25 to about 4 grams per liter, about 0.5 to about 2 grams per liter, about 1 gram per liter to about 5 grams per liter, about 0.75 grams per liter to about 10 grams per liter, about 0.75 grams per liter to about 3 grams per liter, about 0.75 grams per liter to about 2 grams per liter, about 0.75 grams per liter to about 1.5 grams per liter, about 0.5 grams per liter to about 15 grams per liter, about 0.5 grams per liter to about 10 grams per liter, from about 0.5 grams per liter to about 8 grams per liter, from about 0.5 grams per liter to about 6 grams per liter, from about 0.5 grams per liter to about 6 grams per liter, from about 0.1 grams per liter to about 20 grams per liter, from about 0.1 grams per liter to about 10 grams per liter, from about 0.1 grams per liter to about 8 grams per liter, from about 0.1 grams per liter to about 5 grams per liter, from about 0.1 grams per liter to about 3 grams per liter, from about 0.1 grams per liter to about 25 grams per liter, or from 24 grams per liter to about 25 grams per liter.

[0150] In an embodiment, the yield ratio of cytoplasmically produced soluble recombinant crisantaspase to periplasmically produced soluble charge-shielded recombinant crisantaspase fusion protein obtained under similar or substantially similar conditions is from about 1 to about 5. In an embodiment, the yield ratio of cytoplasmically produced soluble charge-shielded recombinant crisantaspase fusion protein to periplasmically produced soluble charge-shielded recombinant crisantaspase fusion protein obtained under similar or substantially similar conditions is at least about 1.

[0151] V. Production of Charge-Shielded Recombinant E. coli Asparagine Fusion Proteins It will be understood by those skilled in the art that a production host strain useful for the method of the present invention can be generated by using a publicly available host cell, such as P. fluorescens MB101, for example, by inactivating the pyrF gene, and / or the type I L-asparaginase gene, and / or the type II L-asparaginase gene, using any of the many suitable methods known in the art and described in the literature. It will also be understood that prototrophy to restore a plasmid can be transformed from strain MB214 to a strain carrying the pyrF gene, such as a plasmid, using any of the many suitable methods known in the art and described in the literature. In addition, in such a strain, the protease can be inactivated and a folding regulator overexpression construct can be introduced using methods well known in the art.

[0152] In an embodiment, a host strain useful for expressing an asparaginase, e.g., E. coli asparaginase type II, in the method of the invention is a Pseudomonas host strain, e.g., P. fluorescens, with a protease deficiency or inactivation (e.g., resulting from a deletion, partial deletion, or knockout) and / or overexpressing a folding regulator, e.g., from a plasmid or bacterial chromosome. In any embodiment, the host strain expresses the auxotrophic markers pyrF and proC, has a protease deficiency, and / or overexpresses a folding regulator. In an embodiment, the host strain expresses any other suitable selection marker known in the art. In any of the above embodiments, the asparaginase, e.g., native type I and / or type II asparaginase, is inactivated in the host strain.

[0153] As described elsewhere herein, an inducible promoter is often used in an expression construct to control expression of the recombinant asparaginase, for example, the lac promoter. In the case of a lac promoter derivative or family member, for example the tac promoter, the effector compound is an inducer, such as a gratuitous inducer such as IPTG (isopropyl-β-D-1-thiogalactopyranoside, also called "isopropylthiogalactoside"). In embodiments, a lac promoter derivative is used and expression of the asparaginase is induced by the addition of IPTG to a final concentration of about 0.01 mM to about 1.0 mM when the cell density reaches a level specified by an OD575 of about 25 to about 160.

[0154] In embodiments, cells are disrupted using equipment for high pressure mechanical cell disruption (commercially available, e.g., Microfluidics microfluidic device, stationary cell disrupter, Niro-Soavi homogenizer, or APV-Gaulin homogenizer). Cells expressing asparaginase are often disrupted, e.g., using sonication. Any suitable method known in the art for lysing cells is used to release the soluble fraction. For example, in embodiments, chemical and / or enzymatic cell lysis reagents, such as cell wall lytic enzymes and EDTA, are often used. The use of frozen or previously stored cultures is also contemplated in the methods herein. Cultures are sometimes OD normalized prior to lysis. For example, cells are often normalized to an OD600 of about 10, about 11, about 12, about 13, about 14, about 15, about 16, about 17, about 18, about 19, or about 20.

[0155] VI. Compositions Comprising Charge-Shielded Proteins Also provided herein is a composition comprising the charge-shielded protein.In some embodiments, the composition is a pharmaceutical composition.In some embodiments, the pharmaceutical composition comprises the charge-shielded protein and one or more pharma- ceutically acceptable carriers.

[0156] Examples of suitable pharmaceutical carriers, excipients, and / or diluents are well known in the art and include phosphate buffered saline solutions, water, emulsions such as oil / water emulsions, various types of wetting agents, sterile solutions, and the like. Compositions containing such carriers can be formulated by well-known conventional methods. Suitable carriers may include any material that, when combined with the bioactive protein of the invention, retains the biological activity of the bioactive protein (see Remington's Pharmaceutical Sciences (1980) 16th edition, Osol, A. Ed). Preparations for parenteral administration may include sterile aqueous or non-aqueous solutions, suspensions, and emulsions). Buffers, solvents, and / or excipients used in the context of pharmaceutical compositions are preferably "physiological" as defined above. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. Parenteral vehicles may include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles may include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives may also be present, including, for example, antimicrobial agents, antioxidants, chelating agents, and inert gases and the like. In addition, the pharmaceutical composition of the present invention may include a proteinaceous carrier, preferably of human origin, such as, for example, serum albumin or immunoglobulin.

[0157] In some embodiments, provided herein are compositions comprising a charge-shielded protein purified protein after a first hydrophobic interaction chromatography column having a purity of up to about 80%, about 85%, about 90%, or about 95%, or greater.

[0158] In some embodiments, provided herein are compositions comprising a charge shielded protein with a purity of at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99%.

[0159] In some embodiments, the composition comprises the charge shielded protein at a concentration of at least 1 mg / mL. In some embodiments, the composition comprises the charge shielded protein at a concentration of at least 5 mg / mL, at least 10 mg / mL, at least 20 mg / mL, at least 50 mg / mL, at least 100 mg / mL, or at least 300 mg / mL. In some embodiments, the composition comprises the charge shielded protein at a concentration of 1-50 mg / mL.

[0160] VI. Treatment Methods In some embodiments, provided herein are methods of treating an individual comprising administering to an individual in need of treatment a composition comprising a charge shielded protein. In some embodiments, the individual has cancer or a neoplastic disease. In some embodiments, the individual has leukemia, lymphoma, or myeloma. In some embodiments, the individual has acute lymphoblastic lymphoma. In some embodiments, the disease is a metabolic disease. In some embodiments, the disease is a hormone deficiency-related disorder, an autoimmune disease, cancer, anemia, neovascular disease, infectious / inflammatory disease, thrombosis, myocardial infarction, or diabetes. EXAMPLES

[0161] The present invention will be more fully understood by referring to the following examples. However, they should not be interpreted as limiting the scope of the present invention. It is understood that the examples and embodiments described herein are for illustrative purposes, and various modifications or changes will be suggested to those skilled in the art in view of them, which are included in the spirit and scope of this application and the scope of the appended claims.

[0162] Example 1. Expression and purification of PAS-asparaginase using ion exchange chromatography as a capture step This example demonstrates expression of a charge-shielded PASylated asparaginase fusion protein (eg, PF745) from periplasmic effluent.

[0163] Recombinant crisantaspase (RC) (asparaginase from Erwinia chrysanthemi) genetically fused at its amino terminus to a 200 amino acid polypeptide sequence (PA200) composed entirely of proline and alanine residues was successfully expressed. The PA200 fusion partner was engineered by XL-Protein GmbH using their proprietary technology, PASylation®, which extends the half-life of biopharmaceuticals by applying intrinsically disordered proteins as a biological alternative to PEGylation. The PA200-RC fusion protein was expressed in Pseudomonas fluorescens to generate recombinant PA200-RC fusion protein that met specific purity and potency targets.

[0164] The fusion protein was named PF745, and construction began with cloning of the PA200-RC DNA fusion in P. fluorescens. An initial screen of 1,040 expression strains at 96-well scale demonstrated successful expression of soluble PA200-RC protein monomer. Strain STR58751 (expressing the periplasmically localized PA200-RC protein) was selected for production of PF745 based on high-titer expression of soluble monomer under multiple fermentation induction conditions, reproducible low N-terminal cleavage profile (<2%), and results from identity, activity, and purity methods. PF745 was expressed and released from cells by osmotic extraction, with selection of periplasmic expression strains during strain engineering. Osmotic extraction was optimized to maximize product release from cells while minimizing host cell contaminant (e.g., host cell protein (HCP)) release.

[0165] As a first chromatographic capture step, we sought to capture PF745 from the periplasmic release by ion exchange chromatography (IEX).

[0166] Anion exchange chromatography (AEX) was performed after osmotic shock extraction of PF745 from STR58751. The extract was adjusted to pH 9 and conductivity of 0.8 mS / cm, loaded onto a POROS 50 HQ AEX column and run in flow-through mode with a loading ratio of 0.82 g paste / mL resin. However, breakthrough of HCPs was observed in fraction 6A1 (lane 14 in Figure 1), indicating that AEX does not provide sufficient enrichment of the target protein (e.g., charge-shielded fusion protein, PF745) when performed as the first purification step.

[0167] Attempts to capture PF745 from a cytoplasmic expressing strain using CEX as an initial capture / purification step were also unsuccessful, and therefore this approach was not subjected to attempts using osmotic shock from STR 58751. Binding to CEX as a second column was also not acceptable, indicating that its use as a capture step would not be successful in enriching the target even in the presence of even higher levels of HCP mixture than the HCP contaminants that the second column would encounter.

[0168] Both the AEX and CEX capture steps showed no capture or very low binding capacity, suggesting that shielding from the PA200 moiety effectively masks the charge on PF745.

[0169] Example 2. Purification of PF745 using size exclusion chromatography followed by CEX Periplasmic extracts from STR58751 were adjusted to 2M ammonium sulfate and loaded onto a Sephacryl S500 resin for size-exclusion chromatography (SEC). Flow-through fractions containing the target molecule were pooled and concentrated using a 100 kDa concentrator, and the concentrated pool was adjusted to 0.5 mS / cm resin and loaded onto a POROS XS cation exchange resin in bind and elute mode. Target binding was observed (see fractions A6-B4, lanes 6-16 in Figure 2), but the binding capacity was low and the majority of the target was observed in the flow-through fraction (lane 3, FT in Figure 2). The load and flow-through fractions were nearly identical, and equal volumes (16 μL) were loaded onto an SDS-PAGE gel. The purity of the target protein in the load was 53.5% as determined by densitometry, and the purity of the target protein in the flow-through was 52.9%, indicating that most of the PF745 protein did not bind to the CEX column and was retained in the flow-through.

[0170] These results indicated that higher loading purity was required for CEX capture of charge-shielded fusion proteins (eg, PF745).

[0171] Example 3. Expression and purification of charge-shielded fusion proteins This example demonstrates the successful purification of a charge-shielded fusion protein (e.g., PF745) from periplasmic effluent. In particular, this example demonstrates that the sequential use of hydrophobic interaction chromatography (HIC), anion exchange chromatography (AEX), and cation exchange chromatography (CEX) increases the purity of PF745 from periplasmic effluent.

[0172] Hydrophobic interaction chromatography Six HIC resins were tested. Toyopearl Butyl-650M showed high binding capacity and acceptable purity and was therefore used as an exemplary HIC column. The osmotic extract was adjusted to 2.5 M NaCl with a final conductivity of 178±15 mS / cm and pH 6.0±0.2. The adjusted periplasmic effluent was filtered and immediately loaded onto a Toyopearl Butyl-650M capture column at a loading ratio of ≦0.17 g paste / mL resin.

[0173] To obtain sufficient protein, the capture column was cycled 8-10 times for each run, for a total of 26 times. The column consistently yielded 8-9 mg of PF745 per gram paste loaded (as measured by A279) with purity values ​​of approximately 75% and 60% as measured by RP-HPLC and SE-HPLC, respectively. SDS-CGE images from a representative HIC step using Butyl-650M resin are shown in Figure 3 and demonstrate the purification obtained by this capture step.

[0174] Anion exchange chromatography It was determined whether the concentrate recovered from ultrafiltration / diafiltration (UF / DF) 1 after HIC could be further purified using an AEX chromatography step to increase purity. POROS HQ resin was used as an exemplary AEX resin. To reduce the risk of potential deamidation, the UF / DF1 recovered concentrate was adjusted to a pH of 9.0±0.2 immediately prior to loading onto the POROS HQ column. Similarly, the collected flow-through and wash pools were also adjusted to a pH of 6.0±0.2 immediately following completion of the POROS HQ chromatography step.

[0175] Each lot was cycled 4-6 times to process all material without exceeding a loading ratio of 5 mg PF745 / mL resin. Regardless of the column cycling, the chromatograms showed that the AEX chromatography was consistent and reproducible. Furthermore, all runs resulted in consistent AEX step yields of 22-25% (Figure 4). Although this yield appears low, it indicates that impurities are being removed rather than product being lost. This is easily demonstrated by comparing the area of ​​the stripping peak with the flow-through + wash product peaks. Additionally, further evidence of impurity removal is shown in the RP-HPLC and SE-HPLC values ​​after this AEX step, which increase to 90% and 80%, respectively. Finally, the HCP content in all AEX eluates was reduced to less than 120 ppm, confirming the HPLC results of increasingly pure samples.

[0176] Therefore, the AEX step performed consistently well and removed a significant amount of impurities after the HIC step.

[0177] Cation exchange chromatography The concentrate recovered from the UF / DF2 step was purified using a CEX chromatography step to test whether it could be used to further increase purity. POROS XS was used as an exemplary CEX resin. Each lot required 3-6 cycles of the CEX step to process all the material without exceeding a loading ratio of 5 mg PF745 / mL resin. Despite cycling the column, the chromatograms showed that the CEX chromatography was consistent and reproducible.

[0178] Furthermore, all runs yielded consistent CEX step recoveries of 48-54%, with products of purity greater than 94% and 100% by RP-HPLC and SE-HPLC, respectively. These purity values ​​were further supported by CGE analysis, which showed that PF745 was effectively separated from impurities remaining in the loading material, resulting in highly pure eluates (Figure 5). Finally, the HCP content of all CEX eluates was less than 3 ppm, confirming the HPLC results of high purity samples.

[0179] Therefore, the CEX step performed consistently well, removing significant amounts of impurities and producing material that exceeded the target purity values, increasing the purity to an even greater extent after the HIC and AEX chromatography steps.

[0180] conclusion The sequential steps of HIC, AEX chromatography, and CEX chromatography, in that order, can be reliably used for the efficient purification of charge-shielded proteins such as PF745 from the periplasmic effluent.

[0181] Example 4. Hydrophobic interaction chromatography for purification of PAS-modified asparaginase This example demonstrates a hydrophobic interaction chromatography (HIC) method for the purification of charge-shielded fusion proteins from periplasmic effluent. In particular, this example demonstrates the use of HIC for HIC resin screening and enrichment of target charge-shielded fusion proteins.

[0182] PF745 (PAS-asparaginase) was expressed and released from cells by osmotic extraction as described, by selection of a periplasmic expression strain during strain engineering. The material was released from the cells, clarified, and capture was tested using hydrophobic interaction chromatography (HIC).

[0183] resin A plate-based resin screen was performed to demonstrate that 13 hydrophobic interaction resins (Table 1) have bind / elute or flow-through purification recovery using 96-well filter plates (Agilent, Cat# 200957-100) and a Biosero Automation System including a Tecan Freedom Evo 200 liquid handling system and a Bionex HiG4 automated centrifuge.

[0184] [Table 1]

[0185] To prepare the resin plates, 50 μL of a 50% slurry of each resin was pipetted into each well of a 96-well plate with a Tecan for 25 μL of target resin per well. High hydrophobic resin plates were prepared with one resin per row in the following order from most hydrophobic to least hydrophobic: Benzyl Ultra, Benzyl, Hexyl-650 C, Capto Phenyl, Butyl-650M, Phenyl-600M, Capto Phenyl ImpRes, and Phenyl Sepharose HP. Low hydrophobic resin plates were prepared with one resin per row in the following order from most hydrophobic to least hydrophobic: Octyl Sepharose 4 FF, Capto Octyl, PPG-600M, Ethyl, and Butyl-650M.

[0186] The plate was centrifuged to filter the slurry liquid. Table 2 includes the chromatography steps starting with stripping the resin with water. The plate was centrifuged after each cycle of pipetting. The resin was then equilibrated with the respective equilibration buffer. The PF745 intermediate from osmotic shock and ultrafiltration / diafiltration (UF / DF) 1 was adjusted with the kosmotrope spike solution to the kosmotrope concentration corresponding to the equilibration buffer. The adjusted PF745 intermediate was then diluted with the corresponding equilibration buffer to 167 mg of paste per mL solution and filtered through a Sartobran P 0.45 / 0.2 μm filter. After loading 150 μL (targeting 1 g of paste per mL resin), the filtrate was collected for flow-through evaluation. The wash and elution filtrates were collected in separate plates as well. The flow-through and elution were analyzed via SDS-CGE.

[0187] [Table 2]

[0188] Figure 6 shows that four resins (Benzyl Ultra, Hexyl-650C, Phenyl-600M, and Capto Phenyl ImpRes) bound to the target under most conditions, as evidenced by very little detectable PF745 band in the flow-through. All resins demonstrated poor binding at 0.25 M sodium sulfate. When PF745 was not bound, the flow-through did not show significantly improved purity with loading.

[0189] The resins identified as having the highest binding based on the flow-through also demonstrated the best elution recovery-Benzyl Ultra, Hexyl-650C, Phenyl-600M, and Capto Phenyl ImpRes (Figure 7). The binding condition resulting in the best recovery was 0.5M ammonium sulfate (triplet "B" in the figure). In addition to promising recovery, the elution showed a significant increase in SDS-CGE purity, evidenced by a reduction in low molecular weight (LMW) bands.

[0190] In contrast, the less hydrophobic resin bound small amounts of PF745 at both 0.25 M sodium sulfate and 0.5 M ammonium sulfate, as evidenced by a PF745 band in the flow-through for these conditions (Figure 8), and in addition, there was no separation of PF745 from impurities. Most conditions yielded low recoveries (Figure 9). Only a 3 M NaCl load in combination with PPG or Butyl-650M resins yielded a significant visible band.

[0191] Benzyl Ultra, Hexyl-650C, Phenyl-600M, and Capto Phenyl ImpRes were tested at 6.1-7.5 mL column scales with the same load material and a load loading of approximately 6 g of paste per mL resin. SDS-CGE images from the Phenyl-600M run demonstrated enrichment of PF745 early in the elution gradient (fractions 1A6-1B5, Figure 10) and separation from eluted impurities (mainly 16-68 kDa) later in the gradient, with a measured load purity of approximately 1% compared to 50-75% in the elution fractions (Figure 10). The load flow-through was not collected, but low and high flow wash fractions showed little breakthrough near the end of the load, suggesting that the majority of the loaded PF745 was bound.

[0192] The elution yields and purity are shown in Table 3. Phenyl-600M showed the most efficient capture properties.

[0193] [Table 3]

[0194] Initial Phenyl-600M and Benzyl Ultra Dynamic Binding Capacity (DBC) testing showed that Benzyl Ultra had greater breakthrough in the flow-through sooner than Phenyl-600M. Phenyl-600M showed binding equivalent to approximately 9.7 g of paste per mL of resin. Phenyl-600M showed 30% higher binding capacity.

[0195] Conditions that were effective for HIC purification of PF745 using the exemplary Phenyl-600M HIC resin included a 0.60 M ammonium sulfate load, which maximized resin capacity. The elution ammonium sulfate concentration was maintained at 0.40 M ammonium sulfate (60-75 mS / cm) to provide high target recovery. The loading and elution pH was set at 5.9 ± 0.1, which consistently captured PF745 material and provided SDS-CGE purity of 40-60% after a single HIC capture step.

[0196] Finally, an exemplary HIC capture method was developed using the exemplary Phenyl-600M resin based on several factors (e.g., (1) EQ, pH of load, wash, and elution, (2) EQ, ammonium sulfate concentration of load, wash, and elution, (3) load addition, and (4) ammonium sulfate concentration of elution). The exemplary HIC method is briefly described in Table 4 and is defined by steps, buffers / solutions, column volumes (CV), and flow rates (cm / h).

[0197] [Table 4]

[0198] conclusion This example shows that HIC chromatography can be used as a capture step to efficiently purify charge-shielded proteins, such as PF745, from periplasmic effluent, achieving greater than 90% purity after a single chromatographic step (e.g., capture step).

[0199] Example 5. Anion exchange chromatography for purification of PAS-modified asparaginase This example demonstrates an anion exchange chromatography (AEX) method for purification of a charge-shielded fusion protein from a cell lysate.

[0200] Anion exchange chromatography was tested as a second chromatography step after the first HIC chromatography step. Five AEX resins (Table 5) were packed into a 0.66 cm Omnifit column (PCE1245-1249) for initial screening.

[0201] [Table 5]

[0202] Figure 11 shows that the flow-through pools of GigaCap Q-650M, POROS XQ, and Super Q-650M had significantly higher SDS-CGE purity (65.6-68.2%) than those of POROS 50 HQ and NH2-750F (32.4-41.0%). Strips from all five runs contained no measurable PF745, indicating high recovery of target and no unintended binding to all tested AEX resins. POROS HQ and NH2-750F flow-through pools had significantly higher HCP levels than the other runs, consistent with the lower SDS-CGE purity (Figure 11). The Super Q-650M flow-through pool had >5x the HCP content of pools from GigaCap Q-650M and POROS XQ. GigaCap Q-650M and POROS XQ produced the highest purity with the lowest HCPs.

[0203] Based on the SDS-CGE integration of the flow-through fractions (FIG. 11), the GigaCap Q-650M flow-through achieved higher purity than POROS XQ and POROS 50 HQ. While the purity of the latter two resins decreased with loading, purity remained relatively consistent across loads for the GigaCap Q-650M. The flow-through pool achieved similar purity results, as shown in Table 6. Thus, all of these resins are suitable for use in the second CEX chromatography step.

[0204] [Table 6]

[0205] No significant differences in RP-HPLC and SE-HPLC purity were observed between runs. The GigaCap Q-650M demonstrated the ability to maintain SDS-CGE purity at a given load, as well as the lowest measured HCP levels.

[0206] GigaCap Q-650M and POROS XQ demonstrated similar performance in terms of SDS-CGE purity of the flow-through fraction (Figure 12). Both resins achieved and maintained high purity <60% for the load, up to 57 mg / mL loading. The rapid drop in purity between 14-16 CV is due to the drop in PF745 concentration as the load transitions to the wash. The GigaCap Q-650M resin maintained slightly higher SDS-CGE purity throughout the load, plus resulted in lower HCP levels than in the previous experiment.

[0207] Screening experiments evaluating five AEX resins demonstrated that GigaCap Q-650M achieved high purification performance. Effective conditions for protein purification by AEX chromatography included load pH 9.0 and 1.0 mS / cm and were robust for load concentrations from 2 to 6 mg / mL. Further experiments demonstrated that a load conductivity of 1.0 mS / cm resulted in high yields without compromising load stability. Conditions favorable for AEX chromatography included load pH of 9.0 ± 0.1, load conductivity of 1.0 ± 0.1 mS / cm, load concentrations from 2 to 6 mg / mL, load held at pH 9 with load additions of 6 to 25 mg / mL for <6 hours, and flow-through titrated to pH 6.0 ± 0.1 within 6 hours.

[0208] Using these conditions for purification, consistent recoveries were obtained with flow-through purities of >85% by RP-HPLC, >80% by SEC-HPLC, HCP levels of <1000 ng / mg, and HCDNA levels of <500 pg / mg. A representative chromatograph of an exemplary AEX chromatography step is shown in Figure 13.

[0209] [Table 7]

[0210] To minimize potential deamidation, it is recommended to perform the loading adjustment to pH 9.0 immediately prior to loading of the AEX and to adjust the flow-through to pH 6.0 as soon as possible after collection.

[0211] conclusion The AEX chromatography step after the initial HIC step improved the purity of the charge-shielded protein PF745.

[0212] Example 6. Cation exchange chromatography for purification of PAS-modified asparaginase This example demonstrates the method of cation exchange chromatography (CEX) as a third chromatographic step for the purification of a charge-shielded fusion protein (eg, PF745) from a cell lysate.

[0213] Cation exchange chromatography was tested as a third chromatographic step for the purification of PF745 after both the HIC and AEX chromatography steps.

[0214] Resin Screening The CEX resin used in the third chromatography step is shown in Table 8.

[0215] [Table 8]

[0216] Four mixed-mode resins (Capto MMC, MX-Trp-650M, CMM HyperCel, Sulfate-650F) were screened to see if they would bind targets (e.g., PF745) at higher conductivity (5-10 mS / cm) for higher resolution and purity. Flow-through purification was performed using a Biosero Automation System, including a 96-well filter plate (Agilent, Cat# 200957-100) and a Tecan Freedom Evo 200 liquid handling system and a Bionex HiG4 automated centrifuge. Figure 14 shows SDS-CGE images of flow-through fractions from eight combinations of pH and conductivity loading subjected to four different mixed-mode resins. The absence of significant PF745 bands in all eight loading conditions in Capto MMC is evidence of good binding. CMM HyperCel demonstrated similar performance, except for breakthrough at pH 5.7 and 20 mS / cm. MX-TRP-650M and Sulfate-650F demonstrated significantly lower binding capacity as evidenced by a significant PF745 band at ≧5 mS / cm.

[0217] Four additional resins were evaluated as third column candidates in the batch mixing study (Capto Core 400, TOYOPEARL NH2-750F, CaPure-HA, and TOYOPEARL PPG-600M). SDS-CGE images of the Capto Core 400 load and flow-through fractions show no significant increase in purity under any condition (Figure 15). Insufficient binding of LMW impurities (<69 kDa) was observed. SDS-CGE images of the NH2-750F load and flow-through fractions show no significant increase in purity under any condition tested (Figure 16). No binding of LMW impurities (<69 kDa) was observed.

[0218] Figure 17 shows SDS-CGE images of CaPure-HA fractions from the batch mix. The flow-through showed no significant PF745 band, measuring near zero concentration, indicating good binding. The absence of a PF745 band in the wash, elution, and stripping fractions indicated that no bound PF745 was recovered. Concentration by UV shows <10% recovery in the elution fraction and negligible recovery in the stripping.

[0219] Finally, FIG. 18 shows the SDS-CGE image of the PPG-600M fraction. The 0.75M ammonium sulfate loading adjustment precipitated PF745 as evidenced by the absence of a band by SDS-CGE. The SDS-CGE acquisition of the 0.25M ammonium sulfate loading condition resulted in a loading purity of 49.6% compared to 55.1% in the flow-through, with the slight increase in purity likely being an artifact of the low concentration in the flow-through fraction, causing the LMW band to fall below the limit of quantification. No significant purity improvement was observed in the flow-through fraction of the 0.25M ammonium sulfate loading condition. At 0.5M ammonium sulfate loading, the lower PF745 band intensity in the flow-through compared to the load indicated significant binding. The stronger intensity in the wash fraction indicated that 0.5M ammonium sulfate did not strongly promote binding and that transition to the wash buffer may elute the protein. In addition, the PF745 band was present in the strip, indicating that it was difficult to achieve good recovery from the 0.5M ammonium sulfate loading condition.

[0220] Three resins, POROS XS, CMM HyperCel, and Capto MMC, were scaled to a 0.66 cm diameter column. Dynamic binding capacity (DBC) studies at 15 mg / mL resin loading showed no significant breakthrough in the flow-through in the chromatograms or SDS-CGE results. Recovery was high (86%) and RP-HPLC purity was consistent with previous results (98.9% purity). These results support protein loading up to 15 mg / mL in CEX chromatography. A 20% safety factor was applied, setting the loading limit at 12 mg / mL. Mixed-mode and gel filtration resin screening experiments demonstrated efficient purification using POROS XS resin.

[0221] Eliminating the pre-elution wash step improved recovery by 20-30% while maintaining product quality comparable to runs with a pre-elution wash. For example, the CEX step using POROS XS resin significantly improved RP-HPLC and SDS-CGE purity and reduced HCP and HCDNA levels.

[0222] DBC runs determined that the load loading could be increased from 5 g / L to 12 g / L with similar purification results. Validated CEX chromatography conditions using a POROS XS as an exemplary column included 1) load conductivity: 1.0 ± 0.1 mS / cm (achieved by UF / DF3), 2) elution NaCl concentration: 8.35 ± 0.08 mM, 3) load loading: ≦12 g / L, and 4) load concentration: ≦6 mg / mL. These conditions are expected to result in recovery ≧70%, RP-HPLC purity ≧97%, and SE-HPLC purity ≧99%. Table 9 shows an exemplary CEX chromatography method followed by UF / DF3 as in FIG. 21.

[0223] [Table 9]

[0224] conclusion A CEX chromatography step was followed after the HIC and AEX steps to further improve the purity of the charge-shielded protein PF745.

Claims

1. A method for purifying a charge-shielded fusion protein from a cell lysate or periplasmic release, the charge-shielded fusion protein comprising a biologically active domain and a charge-shielded domain, and the method comprising hydrophobic interaction chromatography as a first chromatography step.

2. 1. A method for producing a charge-shielded fusion protein from a cell lysate or periplasmic release, said charge-shielded fusion protein comprising a biologically active domain and a charge-shielded domain, said method comprising: i) culturing a cell containing a nucleic acid encoding the charge-shielded fusion protein; ii) purifying the charge-shielded fusion protein, wherein the charge-shielded fusion protein is purified from the cell lysate or periplasmic release using hydrophobic interaction chromatography as a first chromatography step; A method comprising: (a) the charge-shielded fusion protein is at least 45% pure after the first chromatography step. (b) the method further comprises anion exchange chromatography, optionally wherein the anion exchange chromatography is selected from the group consisting of POROS 50HQ resin, POROS XQ resin, and Gigacap Q-650M resin, preferably the anion exchange chromatography is Gigacap Q-650M resin; (c) the method further comprises cation exchange chromatography, optionally i) the cation exchange chromatography is performed on a strong cation exchanger; ii) the cation exchange chromatography is a mixed mode resin; or iii) the cation exchange chromatography is selected from the group consisting of Capto MMC resin, CMM Hypercel resin, Capto SP impres resin, Fracto gel SO 3 -resin, GigaCap S-650S resin, and POROS XS resin, preferably the cation exchange chromatography is POROS XS resin; and / or (d) the method comprises: i) Hydrophobic interaction chromatography; ii) anion exchange chromatography, and iii) Cation Exchange Chromatography The method comprises the steps of: The method according to claim 1 or 2. (a) the biologically active domain is charged at a pH of about 7.0, the charge shielding domain increases the hydrodynamic radius of the protein, and / or the charge shielding domain is uncharged at a pH of about 7.0; (b) the molecular weight of the biologically active domain is less than the molecular weight of the charge shielding domain; (c) the molecular weight of the charge shielding domain is between 10 kDa and 60 kDa; (d) the molecular weight of the charge shielding domain is between 10 kDa and 20 kDa; and / or (e) the molecular weight of the biologically active domain is between 30 kDa and 40 kDa; The method according to any one of claims 1 to 3.

5. 5. The method of any one of claims 1 to 4, wherein the molecular weight of the charge shielded domain is sufficient to increase the in vivo half-life of the charge shielded fusion protein or a multimer of the charge shielded fusion protein, and optionally the in vivo half-life of the charge shielded fusion protein or a multimer of the charge shielded fusion protein is increased compared to the half-life of a protein comprising the biologically active domain without the charge shielded domain or a multimeric protein comprising the biologically active domain without the charge shielded domain. (a) the charge shielding domain has a random coil or disordered structure; and / or (b) the charge shielding domain is a polypeptide consisting of one or more of alanine, serine, and proline residues, optionally the charge shielding domain is a polypeptide consisting of proline and alanine residues; The method according to any one of claims 1 to 5.

7. 1. A method for producing a PASylated biologically active fusion protein from a cell lysate or periplasmic release, comprising: i) culturing a cell containing a nucleic acid encoding the PASylated biologically active protein; ii) purifying the PAS-modified biologically active protein; Including, the PASylated bioactive protein is purified from the cell lysate or periplasmic release using hydrophobic interaction chromatography as a first chromatography step; method.

8. 1. A method for purifying a charge-shielded fusion protein comprising a biologically active domain and a charge-shielded domain from a cell lysate or periplasmic release, comprising the steps of: i) applying a loading solution comprising said charge-shielded fusion protein to a hydrophobic interaction chromatography column; ii) applying a wash solution to the hydrophobic interaction chromatography column; iii) applying an elution solution to the hydrophobic interaction column to elute the charge-shielded fusion protein; iv) applying the eluted charge-shielded fusion protein in iii) as a loading solution to an anion exchange chromatography column; v) eluting the charge-shielded fusion protein from the anion exchange chromatography column; vi) applying the eluted charge-shielded fusion protein in vi) as a loading solution to a cation exchange chromatography column; vii) applying a wash solution to the cation exchange chromatography column; viii) applying an elution solution to the cation exchange chromatography column to elute the charge-shielded fusion protein. A method comprising, in order:

9. (a) The loading solution in step i) is 0.25 to 3M Na ２ SO ４ or 0.25 to 0.6 M NH ４ SO ４ and a pH of 5.5 to 6.5; (b) the elution solution in step iii) comprises 0.3-0.5 M NH 4 SO 4 and has a pH of 5.5-6.5; (c) the loading solution in step iv) has a conductivity of 0.7 to 4.0 mS / cm and a pH of 7.0 to 9.1; (d) the loading solution in step vi) has a pH of 5.9 to 7.0 and a conductivity of 0.7 to 2.5 mS / cm; and / or (e) the elution solution in step viii) has a pH of 6.0 to 7.0 and a conductivity of 0.7 to 4.0 mS / cm; The method according to claim 8.

10. The method according to any one of claims 1 to 9, wherein the hydrophobic interaction chromatography is selected from the group consisting of POROS Benzyl Ultra resin, Hexyl-650C resin, and Phenyl-600M resin, preferably the hydrophobic interaction chromatography is Phenyl-600M resin.

11. the biologically active domain is an asparaginase subunit, and optionally (a) the asparaginase is selected from the group consisting of E. coli asparaginase and Erwinia asparaginase; (b) the asparaginase comprises the amino acid sequence set forth in SEQ ID NO:1, SEQ ID NO:5, or SEQ ID NO:7; or (c) the charge-shielded fusion protein comprises the amino acid sequence set forth in SEQ ID NO:9 or SEQ ID NO:10; The method according to any one of claims 1 to 10.

12. 10. The method of claim 2 or 7, wherein the cell is a bacterial cell, optionally wherein the cell is an E. coli cell or a Pseudomonas cell.

13. A charge-shielded fusion protein produced by the method of any one of claims 1 to 12.

14. 14. A pharmaceutical composition comprising the charge-shielded fusion protein of claim 13 and a pharma- ceutically acceptable carrier.

15. A charge-shielded fusion protein according to claim 13 or a pharmaceutical composition according to claim 14 for use as a medicine.

16. A composition comprising a PAS-ylated asparaginase, wherein the PAS-ylated asparaginase is at least 45% pure.

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