Manufacturing optimization of the multimerized stradomer GL-2045

Abstract

The present disclosure relates to optimization methods for the production of optimally manufactured biologically active proteins called stradomers. The present disclosure further provides compositions and methods useful in the treatment of diseases and conditions, including autoimmune, inflammatory, or infectious diseases. [Selected Figure] Figure 1A

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application No. 62 / 432,402, filed December 9, 2016, the contents of which are incorporated herein by reference in their entirety.

[0002] Description of text files submitted electronically The contents of the text file submitted electronically herein are incorporated herein by reference in their entirety. Computer-readable format copy of Sequence Listing (Filename: GLIK_017_01WO_ST25.txt; Date of recording: December 8, 2017; File size: 15 kb).

[0003] The present invention relates generally to the fields of immunology, autoimmunity, inflammation, and tumor immunology. More specifically, the present invention relates to optimized methods for producing GL-2045. The present invention also relates to novel compositions containing such optimally produced GL-2045, as well as methods for using the GL-2045 compositions. The present invention further relates to the treatment or prevention of pathological conditions, such as autoimmune and inflammatory diseases. [Background technology]

[0004] Pooled human intravenous immunoglobulin (IVIG) has been used to treat immunodeficiencies since the early 1950s and autoimmune and inflammatory diseases in the decades since. IVIG mediates tolerogenic immune effects through several mechanisms, including binding of IVIG aggregates to complement C1q and Fc gamma receptors (FcγR) and crosslinking of these receptors on immune cells such as NK cells (e.g., FcγRIIIa), macrophages (e.g., FcγRIIa), B cells (e.g., FcγRIIb), monocytes, and monocyte-derived cells, including dendritic cells. IVIG is a preparation of sterile, purified immunoglobulin G (IgG) product produced from pooled human plasma, which typically contains more than 90% native IgG, with small and variable amounts of polymeric immunoglobulins, IgA or IgM (Rutter A et al., J Am Acad Dermatol, 2001, June;44(6):1010-1024).

[0005] Substantial published data suggest that the small aggregated IgG fraction of hIVIG, specifically the Fc portion of these aggregates, is overly effective in treating certain diseases mediated by pathological immune complexes. It has been observed that trace amounts (1-5%) of IgG exist in multimeric form within IVIG, and IgG dimers may constitute 5-15% of hIVIG. Alternatives to IVIG therapy have been described that use recombinantly produced Fc multimers that avidly bind Fc receptors and the complement component C1q, similar to IVIG aggregates (see U.S. Patent Application Publication Nos. 2010 / 0239633, 2013 / 0156765, 2015 / 0218236, and International Patent Application Publication No. WO2015 / 132364).

[0006] One such Fc multimer, GL-2045, has been previously disclosed (U.S. Patent Application Publication No. 2013 / 0156765). GL-2045 is a multimerizing, universal stradomer that is a recombinant mimetic of IVIG. GL-2045 binds most or all of the ligands bound by immunoglobulin IgG1 Fc. Furthermore, GL-2045 binds with high affinity and avidity to all standard receptors and to complement C1q, and has 10- to 1,000-fold greater in vitro potency than IVIG. Additionally, GL-2045, or its murine equivalent, is effective in numerous animal models of autoimmune disease, including collagen-induced arthritis, experimental autoimmune neuropathy, idiopathic thrombocytopenic purpura, and experimental autoimmune myasthenia gravis. As such, GL-2045 also has potential clinical utility in the treatment of a wide range of autoimmune diseases, including but not limited to idiopathic thrombocytopenic purpura, chronic inflammatory polyneuropathy, multifocal motor neuropathy, myasthenia gravis, organ transplantation, and rheumatoid arthritis.

[0007] In addition to the advantages of GL-2045 over IVIG in potency and efficacy, GL-2045 demonstrates several advantages in the manufacturing process. IVIG is a pooled human blood product, which means it is obtained from the blood of tens of thousands of donors, whose serum is then mixed together and subsequently purified to remove viruses and other infectious agents, as well as aggregated IgG. As such, availability and supply are limited, and production costs are high. In addition, there is a significant degree of variability between large quantities of IVIG. Conversely, GL-2045 is recombinantly produced, eliminating supply and production cost challenges while providing better control over the manufacturing process.

[0008] GL-2045 homodimers bind to Fc ligands, including Fc gamma receptors and complement C1q, with affinity and without substantial avidity. GL-2045 homodimers also naturally form higher-order multimers that can bind to standard receptors with avidity. GL-2045 homodimers are these higher-order multimers that mimic the enhanced efficacy of IVIG multimer fractions. However, standard cell culture conditions produce variable levels of cell viability, the degree of multimerized protein, and protein titer. Therefore, there is a need in the art for a method for producing GL-2045 that produces a defined multimer pattern, and in particular, a method for producing GL-2045 that produces an increased percentage of higher-order multimers while optimizing cell viability and protein titer. [Prior art documents] [Patent documents]

[0009] [Patent Document 1] US Patent Application Publication No. 2010 / 0239633 [Patent Document 2] US Patent Application Publication No. 2013 / 0156765 [Patent Document 3] US Patent Application Publication No. 2015 / 0218236 [Patent Document 4] International Publication No. 2015 / 132364 [Non-patent literature]

[0010] [Non-Patent Document 1] Rutter A et al.,J Am Acad Dermatol,2001,June;44(6):1010-1024 Summary of the Invention [Means for solving the problem]

[0011] The present invention provides all three advantages over standard manufacturing techniques: improved cell viability, improved high protein titer, and a surprising, substantial increase in the percentage of higher-order multimers. Thus, the optimized manufacturing method provides an optimally manufactured GL-2045 composition with enhanced efficacy for treating inflammatory diseases compared to non-optimally manufactured GL-2045 compositions. Optimized manufacturing of GL-2045 includes optimized upstream manufacturing methods and, in some embodiments, optimized downstream manufacturing methods. Optimized upstream manufacturing methods a) generate high protein titers, b) maintain high cell viability and minimize cell debris, and c) preserve highly ordered multimers of homodimers essential for GL-2045 functionality, and, if desired, both homodimers. Optimized downstream manufacturing methods specifically include various purification techniques used to maintain a selected multimer profile of GL-2045. Thus, in some embodiments, provided herein are GL-2045 compositions with defined multimer profiles.

[0012] In some embodiments, methods are provided for producing GL-2045, comprising culturing Chinese hamster ovary (CHO) cells stably transfected with an expression vector encoding GL-2045 at 37°C ± 1°C until the CHO cells reach a cell density of about 5 million to about 30 million cells / mL, shifting the growth temperature to 37°C ± 1°C to 32.5°C ± 1°C, and harvesting GL-2045 from the medium. In some embodiments, the cells are grown to a density of about 10 million to about 25 million cells / mL before shifting the growth temperature. In some embodiments, the cells are grown to a density of about 10 million to about 15 million cells / mL before shifting the growth temperature. In some embodiments, the cells are grown to a density of about 15 million to about 20 million cells / mL before shifting the growth temperature. In some embodiments, a double temperature shift is used, with a shift from 37°C ± 1°C to 34°C ± 1°C on about day 3 of bioreactor culture, and a second temperature shift from 34°C ± 1°C to 31°C ± 1°C on about day 7.

[0013] In some embodiments, the CHO cells are cultured in ActiCHO P basal medium. In some embodiments, the CHO cells are fed ActiCHO Diet A and ActiCHO Diet B during culture. In some embodiments, the CHO cells are fed every other day. In some embodiments, the expression vector encoding GL-2045 comprises the leader peptide of SEQ ID NO: 1. In some embodiments, the expression vector encoding GL-2045 further comprises a piggyBac transposase recognition sequence and is transfected with a vector encoding piggyBac transposase. In some embodiments, the expression vector encoding GL-2045 produces fewer than 20 genomic insertions.

[0014] In some embodiments, provided is a recombinantly produced GL-2045 made by the methods described herein. In some embodiments, provided is an expression vector encoding GL-2045, comprising a GL-2045 expression cassette, wherein the GL-2045 expression cassette is flanked by piggyBac minimal inverted repeat elements.

[0015] In some embodiments, a method for producing GL-2045 is provided, comprising transfecting CHO cells with an expression vector described herein; culturing the CHO cells in a bioreactor using ActiCHO P medium at a growth temperature of 37°C ± 1°C; feeding the culture of CHO cells daily with Acti CHO Food A and Acti CHO Food B at a growth temperature of 37°C ± 1°C until the culture reaches a cell density of about 10 to about 15 million cells / mL; shifting the growth temperature to 37°C ± 1°C to 32.5°C ± 1°C; and harvesting GL-2045 from the medium, wherein the method produces >80% cell viability at day 21 and a final protein titer of >9,000 mg / mL, wherein >70% of the GL-2045 is present as multimers, and >30% of the multimers are higher order multimeric GL-2045. In some embodiments, cell viability is greater than 95% on day 18 of culture. In some embodiments, the percentage of multimers is greater than 80%.

[0016] In some embodiments, a method for purifying GL-2045 produced by the methods described herein comprises purifying GL-2045 from a culture supernatant by affinity chromatography and purifying GL-2045 by one or more of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography. Polish and

[0017] In some embodiments, depth filtration is used prior to affinity chromatography. In some embodiments, the depth filter is X0HC (Millipore). In some embodiments, the depth filtration unit removes a high percentage of DNA from the supernatant. In some embodiments, the depth filtration unit is an Emphaze™ AEX Hybrid Purifier (3M).

[0018] In some embodiments, affinity chromatography uses a Protein A column. In some embodiments, the Protein A column comprises a NaOH-resistant resin. In some embodiments, the Protein A resin is MabSelect SuRe resin. In some embodiments, affinity chromatography purification involves optimizing purification conditions using one of three different wash buffers. In some embodiments, affinity chromatography purification involves eluting GL-2045 from the affinity chromatography column. In some embodiments, elution of GL-2045 involves elution with a pH gradient. In some embodiments, elution of GL-2045 involves elution without a pH gradient. In one embodiment, elution is performed using a glycine buffer. In another embodiment, elution is performed using an acetate buffer. In some embodiments, the affinity chromatography column is regenerated to remove bound GL-2045. In some embodiments, the affinity chromatography column is regenerated more frequently than suggested by the manufacturer. In some embodiments, the affinity chromatography column is regenerated before each purification cycle. In some embodiments, the affinity chromatography column is regenerated with 0.5 M NaOH buffer.

[0019] In some embodiments, GL-2045 Polish In some embodiments, the anion exchange flow through chromatography comprises using a Q Sepharose fast flow column. In some embodiments, the GL-2045 PolishIn some embodiments, the cation exchange chromatography comprises using a POROS XS column. In some embodiments, the cation exchange chromatography comprises using a sodium acetate elution buffer. In some embodiments, the elution buffer further comprises 36.5-39.0% 1 M NaCl buffer. In one embodiment, the elution method is step elution, and in another embodiment, the elution is gradient elution. In some embodiments, the GL-2045 Polish comprises hydrophobic interaction chromatography. In some embodiments, the hydrophobic interaction chromatography comprises using a butyl FF resin. In some embodiments, the hydrophobic interaction chromatography comprises using a phenyl HP resin. In some embodiments, the hydrophobic interaction chromatography ("HIC") comprises using a phenyl Sepharose 6 fast flow high sub-base resin. In one embodiment, the HIC method is in flow-through mode, and in another embodiment, the HIC method is in binding mode. In some embodiments, the HIC resin results in the isolation of GL-2045 homodimers. In some embodiments, the hydrophobic interaction chromatography comprises using an octyl FF resin. In some embodiments, the column results in the removal of disordered aggregates of GL-2045.

[0020] In some embodiments, a method for purifying GL-2045 is provided, comprising purifying GL-2045 from a culture supernatant by Protein A affinity chromatography, wherein the Protein A column uses an alkaline-tolerant medium such as MabSelect SuRe medium, and the purification is performed with at least two wash cycles, and a clean-in-place (CIP) procedure is performed after each purification run using a high-NaOH regeneration step such as 0.5 M NaOH buffer.

[0021] In some embodiments, a method for purifying GL-2045 comprises purifying GL-2045 by cation exchange chromatography. PolishThis includes using a cation exchange column with a high capacity, high performance column such as POROS XS. separation power The present invention provides a method for purifying GL-2045, comprising eluting the GL-2045 with a resin containing GL-2045, and the elution buffer is a sodium acetate buffer composed of 36.5 to 39.0% 1 M NaCl buffer. In some embodiments, the method for purifying GL-2045 comprises eluting GL-2045 by anion exchange chromatography. Polish In some embodiments, the method further comprises: purifying GL-2045 by hydrophobic interaction chromatography, wherein the anion exchange column contains a strong anion exchange medium that has high chemical stability and allows for cleaning-in-place and sanitation protocols, such as Q Sepharose fast flow medium. Polish wherein the hydrophobic interaction medium is a butyl FF, phenyl HP, or octyl FF resin; and Polish In addition, specific fractions of GL-2045 are selected to be isolated or removed. In some embodiments, GL-2045 is purified and / or Polish The method for producing GL-2045 results in a final protein titer of >4 g / L after all filtration and chromatography steps (i.e., final drug substance). In some embodiments, the final protein composition of GL-2045 contains >70% multimers. In some embodiments, >28% of the multimers are higher order multimers as analyzed by analytical SEC-HPLC.

[0022] In some embodiments, purified GL-2045 produced by the methods described herein is provided. In some embodiments, the purified GL-2045 produced by the methods described herein has a defined multimer pattern that minimizes the percentage of homodimers and / or dimers of homodimers, or otherwise balances the percentage of homodimers, lower order multimers, higher order multimers, and highest order multimers. In some embodiments, methods are provided for treating or preventing an inflammatory, autoimmune, or infectious disease or disorder in a subject in need thereof using the recombinantly produced purified GL-2045 described herein. In some embodiments, the disease or disorder is selected from idiopathic thrombocytopenic purpura, chronic inflammatory polyneuropathy, multifocal motor neuropathy, myasthenia gravis, organ transplant, and rheumatoid arthritis. In some embodiments, GL-2045 is administered intravenously, subcutaneously, orally, intraperitoneally, sublingually, buccally, transdermally, via subcutaneous implantation, or intramuscularly.

[0023] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein the homodimer fraction of the GL-2045 composition comprises less than about 20% of the total composition. In some embodiments, the homodimer fraction comprises 12-19% of the total composition. In other embodiments, the homodimer fraction comprises 14-19% of the total composition. In some embodiments, the homodimer fraction comprises 15.5-17.5% of the total composition. In another embodiment, the homodimer fraction comprises about 16.2% of the total composition.

[0024] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein the dimer fraction of the homodimer of the GL-2045 composition comprises about 7% to about 12% of the total composition. In some embodiments, the dimer fraction of the homodimer comprises about 9% to about 11% of the total composition. In other embodiments, the dimer fraction of the homodimer comprises about 10% of the total composition.

[0025] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein the homodimer trimer fraction of the GL-2045 composition comprises about 5.5% to about 11% of the total composition. In some embodiments, the homodimer trimer fraction comprises about 6.5% to about 8% of the total composition. In other embodiments, the homodimer trimer fraction comprises about 7% of the total composition.

[0026] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein the homodimer tetramer fraction of the GL-2045 composition comprises about 10% to about 16% of the total composition. In some embodiments, the homodimer tetramer fraction comprises about 13% to about 15% of the total composition. In other embodiments, the homodimer tetramer fraction comprises about 14% of the total composition.

[0027] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein the homodimer pentamer fraction of the GL-2045 composition comprises about 6% to about 9% of the total composition. In some embodiments, the homodimer pentamer fraction comprises about 7% to about 8% of the total composition. In other embodiments, the homodimer pentamer fraction comprises about 7% of the total composition.

[0028] In some embodiments, the recombinantly produced GL-2045 composition comprises a hexamer fraction of homodimers comprising about 10% to about 14% of the total composition. In some embodiments, the hexamer fraction of homodimers comprises about 12% to about 13% of the total composition. In other embodiments, the hexamer fraction of homodimers comprises about 12.7% of the total composition.

[0029] In some embodiments, recombinantly produced GL-2045 compositions are provided, wherein the highest order multimers (i.e., in the 7-mer or larger fraction of homodimers) comprise at least about 28% of the total composition. In some embodiments, the highest order multimers comprise no more than 35% of the total composition. In some embodiments, the highest order multimer fraction comprises about 30% to about 34% of the total composition. In other embodiments, the highest order multimer fraction comprises about 31.4% of the total composition.

[0030] In some embodiments, a recombinantly produced GL-2045 composition comprising: (a) the homodimer fraction comprises less than about 20% of the total composition; (b) the highest order multimer fraction comprises at least about 28% of the total composition; (c) the dimer fraction of the homodimer constitutes about 7% to about 12.5% ​​of the total composition; (d) the homodimer trimer fraction comprises about 5.5% to about 11% of the total composition; (e) the homodimer tetramer fraction comprises about 10% to about 16% of the total composition; (f) the homodimer pentamer fraction comprises about 6% to about 10% of the total composition; (g) the homodimer hexamer fraction constitutes about 10% to about 14% of the total fraction; (h) the homodimer dimer to homodimer hexamer fraction constitutes about 40% to about 60% of the total composition; (i) the homodimer trimer to homodimer hexamer fraction constitutes about 32% to about 50% of the total composition; (j) the homodimer tetramer to homodimer hexamer fraction constitutes about 26% to about 39% of the total composition; (k) the homodimer pentamer to homodimer hexamer fraction comprises about 18% to about 23% of the total composition; or (l) Provided is a recombinantly produced GL-2045 composition that is any combination of (a) through (k).

[0031] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein approximately 80% of the total composition comprises higher order multimers, meaning dimers or more of homodimers (i.e., two or more bands). In some embodiments, approximately 60-80% of the total recombinantly produced GL-2045 composition comprises trimers or more of homodimers (i.e., three or more bands). In some embodiments, approximately 54-72% of the total recombinantly produced GL-2045 composition comprises tetramers or more (i.e., four or more bands). In some embodiments, approximately 44-57% of the total composition comprises pentamers or more (i.e., five or more bands). In some embodiments, approximately 38-51% of the total recombinantly produced GL-2045 composition comprises hexamers or more (i.e., six or more bands).

[0032] In some embodiments, recombinantly produced GL-2045 is provided, wherein 2 to 6 bands (i.e., homodimer dimers to homodimer hexamers) of the composition constitute approximately 39 to 61% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, wherein 3 to 6 bands (i.e., homodimer trimers to homodimer hexamers) of the composition constitute approximately 32 to 50% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, wherein 4 to 6 bands (i.e., homodimer tetramers to homodimer hexamers) of the composition constitute approximately 26 to 39% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, wherein 5-6 bands of the composition (i.e., pentamers of homodimers to hexamers of homodimers) comprise approximately 16-23% of the composition. [Brief explanation of the drawings]

[0033] [Figure 1A]Illustrated are GL-2045 fractionation by size exclusion chromatography (FIG. 1A) and analysis of the resulting fractions on a non-reducing gel (FIG. 1B). [Figure 1B] Illustrated are GL-2045 fractionation by size exclusion chromatography (FIG. 1A) and analysis of the resulting fractions on a non-reducing gel (FIG. 1B). [Figure 2] 1 illustrates biolayer interferometry analysis of GL-2045 fractions. [Figure 3A] Illustrated are gel analysis (FIG. 3A) and size exclusion fractionation results (FIG. 3B) for GL-2045. [Figure 3B] Illustrated are gel analysis (FIG. 3A) and size exclusion fractionation results (FIG. 3B) for GL-2045. [Figure 4A] 1 illustrates the effect of GL-2045 fractions in a complement-dependent cell killing assay. [Figure 4B] 1 illustrates the effect of GL-2045 fractions in a complement-dependent cell killing assay. [Figure 4C] 1 illustrates the effect of GL-2045 fractions in a complement-dependent cell killing assay. [Figure 4D] 1 illustrates the effect of GL-2045 fractions in a complement-dependent cell killing assay. [Figure 5A] Illustrated are the elution chromatogram (FIG. 5A) and SDS-Page analysis (FIGS. 5B and 5C) of GL-2045 for use in an FcγRIIIa binding assay. [Figure 5B] Illustrated are the elution chromatogram (FIG. 5A) and SDS-Page analysis (FIGS. 5B and 5C) of GL-2045 for use in an FcγRIIIa binding assay. [Figure 5C] Illustrated are the elution chromatogram (FIG. 5A) and SDS-Page analysis (FIGS. 5B and 5C) of GL-2045 for use in an FcγRIIIa binding assay. [Figure 6A] 6 illustrates the binding of the eluted fractions shown in FIG. 5 to FcγRIIIa (FIG. 6A) and the best fit curve (FIG. 6B). [Figure 6B]6 illustrates the binding of the eluted fractions shown in FIG. 5 to FcγRIIIa (FIG. 6A) and the best fit curve (FIG. 6B). [Figure 7] SDS-Page analysis of anion exchange fractions is illustrated: GL-GLM-01 = recombinant, unfractionated Fc (G001), GL-GLM-02 = unfractionated GL-2045, GL-GLM-05 = GL-2045 fractionated at pH 6.0, GL-GLM-06 = GL-2045 fractionated at pH 6.5, GL-GLM-07 = GL-2045 fractionated at pH 7.0, GL-GLM-08 = GL-2045 fractionated at pH 7.5. [Figure 8] Illustrated are the results of a neutrophil chemotaxis assay using C5a as a chemoattractant in the presence of unfractionated fractionated GL-2045: GL-GLM-01 = recombinant, unfractionated Fc (G001), GL-GLM-02 = unfractionated GL-2045, GL-GLM-05 = fractionated GL-2045 at pH 6.0, GL-GLM-06 = fractionated GL-2045 at pH 6.5, GL-GLM-07 = fractionated GL-2045 at pH 7.0, GL-GLM-08 = fractionated GL-2045 at pH 7.5. [Figure 9] Illustrates the cell density (in millions / mL) of CHO cells grown in a panel of different media on days 4, 8, and 10 of culture. [Figure 10] Illustrates the % cell viability of CHO cells grown in a panel of different media on days 4, 8, and 10 of culture. [Figure 11] 1 illustrates the protein titer (mg / mL) of CHO cells grown in a panel of different media at day 10 of culture. [Figure 12A] 1 illustrates a gel analysis of GL-2045 protein produced from CHO cells grown in the panel of media illustrated in FIGS. 9-11. [Figure 12B] 1 illustrates a gel analysis of GL-2045 protein produced from CHO cells grown in the panel of media illustrated in FIGS. 9-11. [Figure 13A]Exemplary feeding schedules are shown for PowerCHO3 CD, ADCF-Mab Hyclone, and ActiCHO P media (FIG. 13A), and for Cellvento, BalanCD CHO Growth A, CD FortiCHO Life, and CD4MCHO Hyclone media (FIG. 13B). [Figure 13B] Exemplary feeding schedules are shown for PowerCHO3 CD, ADCF-Mab Hyclone, and ActiCHO P media (FIG. 13A), and for Cellvento, BalanCD CHO Growth A, CD FortiCHO Life, and CD4MCHO Hyclone media (FIG. 13B). [Figure 14] Illustrates the cell density (e6 cells / mL) of CHO cells grown in a panel of different media+feed combinations from days 0 to 11 of culture. [Figure 15] Illustrates the % cell viability of CHO cells grown in a panel of different media+feed combinations from days 0 to 11 of culture. [Figure 16] Illustrates the protein titer (mg / mL) of GL-2045 from CHO cells grown in a panel of different media+feed combinations from days 0 to 11 of culture. [Figure 17] 13A-13B illustrate SDS-PAGE analysis of the effect of medium+feeding combinations and schedules illustrated in FIGS. 13A-13B on GL-2045 multimerization. [Figure 18A] Illustrated are the effects of daily ActiCHO-P medium + feeding (red) and every other day ActiCHO-P medium + feeding (blue) on cell density (FIG. 18A), cell viability (FIG. 18B), culture pH (FIG. 18C), and GL-2045 protein titer (FIG. 18D). [Figure 18B]Illustrated are the effects of daily ActiCHO-P medium + feeding (red) and every other day ActiCHO-P medium + feeding (blue) on cell density (FIG. 18A), cell viability (FIG. 18B), culture pH (FIG. 18C), and GL-2045 protein titer (FIG. 18D). [Figure 18C] Illustrated are the effects of daily ActiCHO-P medium + feeding (red) and every other day ActiCHO-P medium + feeding (blue) on cell density (FIG. 18A), cell viability (FIG. 18B), culture pH (FIG. 18C), and GL-2045 protein titer (FIG. 18D). [Figure 18D] Illustrated are the effects of daily ActiCHO-P medium + feeding (red) and every other day ActiCHO-P medium + feeding (blue) on cell density (FIG. 18A), cell viability (FIG. 18B), culture pH (FIG. 18C), and GL-2045 protein titer (FIG. 18D). [Figure 19] Illustrates the effect of daily ActiCHO-P medium plus feeding (red) and every 3 days ActiCHO-P medium plus feeding (blue) on GL-2045 protein titer. [Figure 20A] 20A illustrates the effect of combining PowerCHO2 (red) basal medium with ActiCHO Diet A on cell viability (FIG. 20A) and GL-2045 protein titer (FIG. 20B) compared to using ActiCHO Diet A (blue) with ActiCHO-P basal medium. [Figure 20B] 20A illustrates the effect of combining PowerCHO2 (red) basal medium with ActiCHO Diet A on cell viability (FIG. 20A) and GL-2045 protein titer (FIG. 20B) compared to using ActiCHO Diet A (blue) with ActiCHO-P basal medium. [Figure 21A] 21A illustrates the effect of optimized shake flask conditions on cell density (FIG. 21A), cell viability (FIG. 21B), and GL-2045 protein titer (FIG. 21C). [Figure 21B]21A illustrates the effect of optimized shake flask conditions on cell density (FIG. 21A), cell viability (FIG. 21B), and GL-2045 protein titer (FIG. 21C). [Figure 21C] 21A illustrates the effect of optimized shake flask conditions on cell density (FIG. 21A), cell viability (FIG. 21B), and GL-2045 protein titer (FIG. 21C). [Figure 22] 1 illustrates SDS-PAGE analysis of Protein A purified GL-2045. [Figure 23A] Illustrated is the elution profile from a Protein A column after elution of GL-2045 by pH gradient elution (FIG. 23A) and SDS-PAGE analysis of isolated fractions (FIG. 23B). [Figure 23B] Illustrated is the elution profile from a Protein A column after elution of GL-2045 by pH gradient elution (FIG. 23A) and SDS-PAGE analysis of isolated fractions (FIG. 23B). [Figure 24] Elution chromatograms and non-reducing SDS-PAGE analysis are illustrated. [Figure 25] Elution chromatograms and non-reducing SDS-PAGE analysis are illustrated. [Figure 26] The elution chromatographs of runs C1 to C3 are shown as examples. [Figure 27] Non-reducing SDS-PAGE analysis of elution peaks 38%-39% from runs C1-C3 is illustrated. [Figure 28] 1 illustrates the densitometry analysis of ion chromatography purified GL-2045. [Figure 29A] Illustrated is the elution profile of the HIC column (top panel) and SDS-PAGE analysis of the elution and flow-through fractions (FT) (bottom panel). [Figure 29B] Illustrated is the elution profile of the HIC column (top panel) and SDS-PAGE analysis of the elution and flow-through fractions (FT) (bottom panel). [Figure 30] Illustrated is the elution profile from HIC column polishing of M045 (right panel) and Nu-PAGE analysis (right panel). [Figure 31] 1 shows an example of the elution profile from HIC column polishing of M045. [Figure 32] 1 illustrates the elution profile and SDS-PAGE analysis of M045 from HIC column polishing. [Figure 33] 1 illustrates the defined multimer pattern of an optimally prepared GL-2045 composition. DETAILED DESCRIPTION OF THE INVENTION

[0034] The approaches to producing optimized recombinant GL-2045 described herein include optimized upstream manufacturing methods that result in enhanced GL-2045 multimerization while optimizing cell viability and protein titer. In some embodiments, optimized downstream manufacturing achieves optimized conditions for the pharmaceutical agent. Additionally, provided herein are compositions comprising GL-2045 with defined multimerization patterns. The compositions provided herein have utility for treating autoimmune diseases, inflammatory diseases, allergies, antibody-mediated diseases, and complement-mediated diseases.

[0035] As used herein, "drug substance" refers to the final dosage form of GL-2045 as sold by the manufacturer.

[0036] As used herein, the use of the word "a" or "an" when used in conjunction with the word "comprising" in the claims and / or specification may mean "one," but is also consistent with the meaning of "one or more," "at least one," and "one or more than one."

[0037] As used herein, the terms "biomimetic," "biomimetic molecule," "biomimetic compound," and related terms refer to an artificial compound that mimics the function of another compound, such as pooled human intravenous immunoglobulin ("hIVIG"), a monoclonal antibody, or the Fc fragment of an antibody. "Biologically active" biomimics are compounds that have the same or similar biological activity as their naturally occurring counterparts. "Naturally occurring" refers to a molecule or portion thereof that is normally found in an organism. "Naturally occurring" also means substantially occurring in nature. "Immunologically active" biomimetics are biomimics that exhibit the same or similar immunological activity as naturally occurring immunologically active molecules, such as antibodies, cytokines, interleukins, and other immunological molecules known in the art. In a preferred embodiment, the biomimetics of the present invention are optimized multimerizing stradomers, as defined herein (e.g., optimally engineered GL-2045).

[0038] "Directly linked" means two sequences joined together without any intervening or extraneous sequence, e.g., an amino acid sequence resulting from the insertion of a restriction enzyme recognition site into DNA or a cloning fragment. Those skilled in the art will understand that "directly linked" encompasses the addition or removal of amino acids, so long as the ability to multimerize is not substantially affected.

[0039] "Homologous" refers to identity across the entire sequence of a given nucleic acid or amino acid sequence. For example, "80% homologous" means that the given sequence shares approximately 80% identity with the claimed sequence and may contain insertions, deletions, substitutions, and frameshifts. Those skilled in the art will understand that sequence alignment can be performed to take into account insertions and deletions and determine identity across the entire length of the sequence.

[0040] It has been described that hIVIG binds to and completely saturates neonatal Fc receptors (FcRn) and that such competitive inhibition of FcRn may play an important role in the biological activity of hIVIG (e.g., F. Jin et al., Human Immunology, 2005, 66(4)403-410). Because immunoglobulins that strongly bind to Fcγ receptors also bind to FcRn, at least to some extent, those skilled in the art will recognize that stradomers capable of binding to more than one Fcγ receptor may also bind to and completely saturate FcRn.

[0041] There are two human polymorphs of IgG1, called the DEL polymorph and the EEM polymorph. The DEL polymorph has a D at position 356 and an L at position 358, and the EEM polymorph has an E at position 356 and an M at position 358 (Kabat numbering, SEQ ID NOS: 2 and 3, EEM and DEL polymorphs, respectively). The stradomers provided herein can contain either the DEL polymorph or the EEM IgG1 polymorph. Thus, even though text about a particular variant is made explicitly in the context of the DEL polymorph, one of skill in the art will understand that the same mutations can be made to the EEM polymorph to produce the same result.

[0042] US2010 / 0239633 discloses the use of linked immunoglobulin Fc domains to create ordered multimerized immunoglobulin Fc biomimetics of hIVIG (biologically active ordered multimers known as stradomers) that contain short sequences containing restriction sites and affinity tags between the individual components of the stradomer for the treatment of pathological conditions, including autoimmune diseases and other inflammatory conditions. See US2010 / 0239633, which is incorporated by reference in its entirety. US2013 / 0156765 discloses stradomers in which the individual components are directly linked rather than separated by restriction sites or affinity tags. US2013 / 0156765 also specifically discloses a multimerizing stradomer (GL-2045) comprising an IgG1 Fc domain with an IgG2 hinge multimerization domain directly linked to its C-terminus, which exhibits improved multimerization and complement fixation relative to N-terminally linked constructs (e.g., GL-2019 described in US2010 / 0239633). See US2013 / 0156765, which is incorporated by reference in its entirety. The structures of GL-2045 are provided as SEQ ID NOs: 4 and 5 (EEM and DEL polymorphs, respectively).

[0043] Stradomer unit monomer As used herein, the term "stradomer unit monomer" refers to a single contiguous peptide molecule that, when associated with at least a second stradomer unit monomer, comprises at least one Fc domain and, in the case of GL-2045 and an IgG2 hinge multimerization domain, forms a homodimeric "stradomer unit." In a preferred embodiment, a GL-2045 stradomer unit is composed of two associated stradomer unit monomers. However, a GL-2045 stradomer may also contain more than two stradomer unit monomers.

[0044] The optimally engineered stradomer of the present invention (optimally engineered GL-2045) contains a direct linkage between the N-terminus of the IgG1 Fc monomer and the C-terminus of the leader peptide (SEQ ID NO: 1) and the C-terminus of the IgG1 Fc and the N-terminus of the multimerization domain IgG2 hinge (SEQ ID NO: 6).

[0045] For clarity, those skilled in the art will understand that optimally engineered stradomer molecules of the present invention can be constructed by preparing polynucleotide molecules encoding Fc domain monomers and multimerization regions. Such polynucleotide molecules can be inserted into expression vectors, which can be used to transform bacterial populations or transfect mammalian cell populations. Stradomer unit monomers can then be produced by culturing the transformed bacteria or transfected mammalian cells under appropriate culture conditions. For example, by selecting cells with geneticin / G418, clonal cell lines can be achieved that continue to pool stably transfected cells. Alternatively, cells can be transiently transfected with DNA encoding optimally engineered stradomers of the present invention (e.g., DNA encoding a stradomer according to SEQ ID NO: 4 or 5) under the control of a CMV promoter. The expressed stradomer unit monomers can then form functional stradomer units and stradomers either upon self-assembly of stradomer monomers or units using the interconnection of stradomer monomers or upon association of stradomer monomers. The expressed stradomers can then be purified from the cell culture medium by downstream manufacturing methods described herein (e.g., affinity chromatography, ion exchange chromatography, and / or hydrophobic interaction chromatography). One of skill in the art will understand that the leader peptide included in the nucleic acid construct is used to facilitate the production of the stradomer unit monomer peptides only and is cleaved upon expression of the mature protein. Thus, the biologically active biomimetics of the present invention do not include a leader peptide.

[0046] Cluster Stradomar In one embodiment, the optimally manufactured GL-2045 produced according to the present disclosure is a cluster stradomer. A "cluster stradomer" is a biomimetic with a radial configuration, including a central "head" and two or more "legs," each containing one or more Fc domains capable of binding at least one Fc gamma receptor and / or complement. Cluster stradomers are also known as "multimerization stradomers" due to the presence of multimerization domains that result in multimerization of the stradomer. Thus, a serial stradomer containing multiple Fc domains on a single stradomer monomer molecule can still be classified as a cluster stradomer or multimerization stradomer, as long as the molecule also contains at least one multimerization domain. Each cluster stradomer is composed of more than one homodimeric protein, each referred to as a "cluster stradomer unit." Each cluster stradomer unit is composed of at least one region that multimerizes at least one functional Fc domain and a "leg" region that includes at least one functional Fc domain. Once multimerized to another cluster stradomer unit, the multimerization region creates the "head" of the cluster stradomer. The leg regions may be capable of binding as many complement molecules as there are Fc domains in each leg region. For example, the leg regions may be capable of binding as many C1q molecules as there are Fc domains in each leg region. Thus, cluster stradomers are biomimetic compounds that can bind more than one C1q molecule, thereby preventing complement-mediated lysis, also known as complement-dependent cytotoxicity (CDC).

[0047] The multimerization region contained within the optimally engineered stradomers of the present invention is the IgG2 hinge region. As is known in the art, the hinge region of human IgG2 can form covalent dimers (Yoo, EM et al. J. Immunol. 170, 3134-3138 (2003), Salfeld Nature Biotech. 25, 1369-1372 (2007)). IgG2 dimerization is potentially mediated through the IgG2 hinge structure by a C-C bond (Yoo et al. 2003), suggesting that the hinge structure alone may mediate dimerization. However, the amount of IgG2 dimers found in human serum is limited. It is estimated that the amount of IgG2 present as a homodimer is less than 10% of the total IgG2 (Yoo et al. 2003). Furthermore, there is no quantitative evidence of IgG2 multimerization beyond homodimer dimers. (Yoo et al. 2003). That is, native IgG2 has not been found to form higher order multimers in human serum. IgG2 hinge-containing stradomers (e.g., optimally engineered GL-2045) exist as higher order multimers, and unlike native IgG2 in human serum, where the IgG2 hinge interactions are variable and dynamic, GL-2045 has been demonstrated to form highly stable multimers as evidenced by non-reducing SDS-PAGE gels, analytical ultracentrifugation, and a 3-month stability study at 37°C and 100% humidity. Furthermore, it is surprising that the amount of multimers in IgG2 hinge-containing stradomer-formulated drugs significantly exceeds the approximately 10% dimers observed for IgG2 in human serum, and does not contain any multimers. For example, the proportion of stradomers that are multimers, including dimers, trimers, tetramers, and higher order multimers of homodimers, is greater than 20%, and may be greater than 30%, 40%, 50%, 60%, 70%, 80%, or even 90%. In particularly preferred embodiments, the proportion of GL-2045 that exists as homodimers is 10-20%, and the corresponding proportion of GL-2045 that exists as highly ordered multimers of homodimers is greater than 70%.

[0048] The amino acid sequence of GL-2045 is set forth in SEQ ID NOs:4 and 5.

[0049] As used herein, the term "isolated" polypeptide or peptide refers to a polypeptide or peptide that has no naturally occurring counterpart or that has been separated or purified from components that naturally accompany it, for example, in tissues such as the pancreas, liver, spleen, ovaries, testes, muscle, joint tissue, nervous tissue, gastrointestinal tissue, or breast tissue or tumor tissue (e.g., breast cancer tissue), or in bodily fluids such as blood, serum, or urine. Typically, a polypeptide or peptide is considered "isolated" when it is at least 70% by dry weight free from naturally associated proteins and other naturally occurring organic molecules. Preferably, preparations of the polypeptide (or peptide) of the invention are at least 80%, more preferably at least 90%, and most preferably at least 99% by dry weight of the polypeptide (peptide) of the invention. Because chemically synthesized polypeptides or peptides are essentially separated from components that naturally accompany them, synthetic polypeptides or peptides are "isolated."

[0050] An isolated polypeptide (or peptide) of the invention can be obtained, for example, by expression of a recombinant nucleic acid encoding the polypeptide or peptide or by chemical synthesis. A polypeptide or peptide produced in a cell line different from the source in which it naturally occurs is "isolated" because it does not necessarily contain components that naturally accompany it. In a preferred embodiment, an isolated polypeptide of the invention contains only the sequence corresponding to the IgG1 Fc monomer and IgG2 hinge multimerization domain (SEQ ID NO: 6) and does not contain additional sequences that may aid in cloning or purification of the protein (e.g., introduced restriction enzyme recognition sites or purification tags). The degree of isolation or purity can be measured by any appropriate method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0051] Manufacturing method GL-2045 forms ordered homodimers and is active in all homodimer and multimer fractions. It is essential for GL-2045 function that the manufacturing process produces an optimized multimer profile. As used herein, "optimized multimer profile" or "optimized multimerization profile" refers to a combination of GL-2045 homodimers and highly ordered multimers that produces a desired biological outcome for GL-2045 as an IVIG mimetic (e.g., without being bound by theory, improved binding to C1q by initial activation of the complement system and / or subsequent inhibition of complement activation, and prevention of CDC, at the level of C3 / C3b). Those skilled in the art will recognize that it may be beneficial to isolate various multimer fractions from optimally manufactured GL-2045 as separate products, either alone or in combination with other factors, including for other therapeutic purposes. For example, as provided in the Examples, larger multimer fractions of GL-2045 are more active than smaller multimer fractions in binding to C1q, regulating downstream complement-mediated effector functions, and binding low-affinity FcγRs. Thus, the methods of the present invention are directed not only to GL-2045 compositions comprising optimized multimer profiles, but also to GL-2045 compositions that involve only selecting multimers based on desired effector function. In such embodiments, an optimized multimer profile of GL-2045 that produces one desired biological outcome may differ from an optimized multimer profile that produces another desired biological outcome.

[0052] Without being bound by theory, it is believed that homodimers function as receptor and ligand buffers similar to unaggregated IgG1. Higher-order multimers bind with increased avidity to low-affinity Fcγ receptors and complement factors (e.g., hexamer C1q) and, as described herein, demonstrate enhanced biological potency when compared to homodimers or lower-order multimers (e.g., dimers, trimers, and / or tetramers of GL-2045 homodimers). Therefore, the degree of multimerization is an essential upstream and downstream manufacturing consideration in the production of clinically effective GL-2045. Thus, it is desirable not only to maintain optimal cell viability, high protein titer, and an optimal multimerization profile of GL-2045 through optimized upstream manufacturing methods, but also to maintain and / or improve the optimal multimerization profile of GL-2045 through optimized downstream manufacturing methods.

[0053] In some embodiments, the optimized production methods described herein result in GL-2045 protein compositions in which at least 70% or at least 80% of the GL-2045 exists as non-homodimers (e.g., dimers of homodimers, trimers of homodimers, etc.). In some embodiments, more than 70% or more than 80% of the GL-2045 exists as non-homodimers. For example, optimized production methods may result in GL-2045 protein compositions in which 80%, 85%, 90%, 95% or more of the GL-2045 exists as non-homodimers. In some embodiments, the protein composition comprises at least 28% or at least 30% of the GL-2045 present as highest order multimers (i.e., homodimeric 7-mers or greater). In some embodiments, the protein composition comprises no more than 35% of the GL-2045 present as highest order multimers. In some embodiments, the protein composition is comprised of at least about 35% of GL-2045 present as tetramers, pentamers, hexamers, and heptamers (i.e., at least 35% of the total GL-2045 composition is comprised of 4-6 fractions). In some embodiments, at least about 35% of GL-2045 is present as trimers or higher of homodimers (i.e., at least 35% of the total GL-2045 composition is comprised of 3-6 fractions). In some embodiments, at least about 35% of GL-2045 is present as trimers, tetramers, pentamers, or hexamers of homodimers (i.e., at least 35% of the total GL-2045 composition is comprised of 3-6 fractions). In some embodiments, at least about 35% of GL-2045 is present as tetramers or higher of homodimers (i.e., at least 35% of the total GL-2045 composition is comprised of 4-6 fractions). In some embodiments, at least about 35% of the GL-2045 exists as tetramers and pentamers of homodimers (i.e., at least 35% of the total GL-2045 composition is composed of 4 and 5 fractions), and in some embodiments, at least about 35% of the GL-2045 exists as pentamers or higher of homodimers (i.e., at least 35% of the total GL-2045 composition is composed of 5 or more fractions).In some embodiments, at least about 35% of the GL-2045 exists as pentamer and hexamer homodimers (i.e., at least 35% of the total GL-2045 composition is composed of 5- and 6-mer fractions). In some embodiments, at least about 35% of the GL-2045 exists as hexamer or higher homodimers (i.e., at least 35% of the total GL-2045 composition is composed of 6-mer or higher fractions). In some embodiments, at least about 35% of the GL-2045 exists as 7-mer or higher homodimers (i.e., at least 35% of the total GL-2045 composition is composed of 7-mer or higher fractions). For example, the optimized production methods described herein can result in GL-2045 protein compositions in which 40%, 45%, 50%, 55% or more of the GL-2045 exists as pentamer or higher homodimers. Current manufacturing methods for Fc-containing therapeutics (e.g., monoclonal antibodies) have focused on increasing protein titer and yield through downstream filtration steps. These methods generally do not consider the effect of the manufacturing process on the multimerization of Fc-containing proteins and, in stark contrast to the methods described herein, aim to minimize protein aggregation and multimerization. Surprisingly, the culture conditions that produce the highest protein yield of GL-2045 do not necessarily produce the highest percentage of GL-2045 present as multimers. Thus, the data described herein demonstrate that manufacturing variables that affect overall protein titer are, at least in part, independent of variables that affect the multimerization profile. Therefore, one skilled in the art would not be able to predict that upstream manufacturing conditions will affect GL-2045 multimerization based on the current state of the art.

[0054] For example, protocols developed for recombinant protein production using Chinese hamster ovary (CHO) cells provide for a temperature shift from 37°C to 31°C on specific days of culture (Ouguchi et al., Cytotechnology, 52(3), pp. 199-207, (2006); Masterson and Smales, Pharmaceutical Bioprocessing, 2(1), pp. 49-61, (2014)). However, in contrast to that described by Ouguchi et al., we have found that a temperature shift from 37°C to 32.5°C results in maintaining cell density and high cell viability while optimizing for protein titer. Furthermore, we have found that shifting the temperature based on cell density, rather than based on a given day of culture as previously described, results in an increase in GL-2045 protein titer of approximately 10 g / L.

[0055] Furthermore, the inventors have discovered that maintaining an optimized multimerization profile resulting from an optimized upstream manufacturing process depends in part on optimized downstream manufacturing processes (e.g., affinity chromatography and / or ion exchange chromatography). Filtration and purification techniques for monoclonal antibodies (mAbs) and Fc fusion proteins have been widely described and are commonly used. However, when applied to GL-2045, these techniques result in unpredictable alterations of the GL-2045 multimerization profile. For example, the inventors have surprisingly discovered that most Protein A columns are not suitable for purifying GL-2045, despite their routine use in mAb and Fc fusion protein purification, as demonstrated in Example 8. Protein A is a very expensive reagent, costing millions of dollars for use according to Good Manufacturing Practice (GMP) purification of a single drug, and must be reused more than 100 times to be economically viable. Like mAbs and Fc fusion proteins, GL-2045 binds to Protein A, but unlike mAbs and Fc fusion proteins, GL-2045 does not completely dissociate from Protein A during normal elution steps due to the avid binding of GL-2045 to Protein A. The inventors unexpectedly discovered that utilizing a Protein A column for purification of GL-2045, where an optimal multimerization profile is maintained, requires a more frequent column cleaning schedule.

[0056] The present inventors further discovered that the protein A column clean-in-place (CIP) procedure commonly used in the art unexpectedly results in altered GL-2045 multimerization profiles. Typical CIP procedures entail column cleanup at the end of a purification run, which can involve multiple cycles of passing the protein supernatant through the column. However, the present inventors discovered that the high binding avidity of GL-2045 results in a lack of dissociation of GL-2045 from protein A. As a result, the binding site of protein A remains occupied, preventing GL-2045 binding in subsequent cycles and resulting in the loss of homodimers. Thus, the present inventors unexpectedly discovered that, in contrast to protocols utilized with mAbs or Fc-fusion proteins, CIP cleanup of a protein A column with GL-2045 must be performed more frequently than with monoclonal antibodies or Fc-fusion proteins, and with highly stringent regeneration buffers such as 0.5 M NaOH.

[0057] pH elution gradients are commonly used with Protein A columns to optimize protein yield during purification. The present inventors surprisingly discovered that pH elution gradients used in the art to optimize Protein A column yields of monoclonal antibodies or Fc fusion proteins can cause the desired loss of homodimeric or higher-order multimeric components of GL-2045, thus potentially altering the multimerization profile of GL-2045 (as demonstrated in Example 10). Thus, the present inventors also determined a means to use elution gradient techniques to optimize the combination of GL-2045 yield and multimerization. In addition, the present inventors surprisingly discovered that pH elution gradients can be applied to Protein A columns for a novel purpose: to separate distinct, maximally highly ordered GL-2045 multimers from homodimeric aggregates (as demonstrated in Example 11).

[0058] The present inventors have also discovered that when using ion exchange columns commonly used in the art to purify monoclonal antibodies and Fc fusion proteins, such as anion exchange and cation exchange, changes in pH and / or salt can alter the multimerization profile of GL-2045, as demonstrated in Example 12. This is in stark contrast to monoclonal antibodies or Fc fusion proteins, where changes in salt or pH can result in small amounts of protein loss but do not alter the composition of the drug. Additionally, the present inventors surprisingly discovered that salt and / or pH adjustments can be used for novel purposes to separate the most highly ordered GL-2045 multimers from homodimeric aggregates that may be composed of similar molecular masses. As demonstrated in Example 13, the present inventors use functional assays to demonstrate that removal of disordered aggregates from the highest-order multimer fraction is associated with higher potency and a more highly purified GL-2045 product.

[0059] Hydrophobic interaction (HIC) columns are used for high-yield capture of monoclonal antibodies. Polish HIC columns are commonly used in the art to purify monoclonal antibodies and Fc fusion proteins through a variety of mechanisms, including removal of smaller species from full-length forms, separation of active from inactive forms, and viral clearance. However, when used in the context of GL-2045, the inventors found standard HIC columns to be unpredictable. As demonstrated in Example 14, seven HIC columns composed of different matrices were associated with widely different capture rates, ranging from 16% to 62%, despite the same supernatant and the same buffer being used with all columns.

[0060] Furthermore, we predict that changing the buffer may alter the multimerization profile of GL-2045, which may result in the loss of small amounts of protein or a more incomplete multimerization. PolishThis is in stark contrast to monoclonal antibodies or Fc fusion proteins, which may result in a change in the underlying composition of the drug but may not result in a change in the underlying composition of the drug.

[0061] Furthermore, the present inventors discovered that because the GL-2045 homodimer is composed of an IgG1 Fc and multimerization domains that allow it to form highly ordered multimers, GL-2045 avidly binds to all or nearly all of the many ligands and targets that native IgG1 Fc homodimers bind to without binding affinity. This surprisingly includes all low-affinity Fc receptors and complement C1q, as well as protein A and protein G, which are commonly used in purification columns. However, this avid binding also results in more undesirable potential target binding, such as endotoxin. For this reason, the present inventors conducted purification of GL-2045 with protein A and purification of protein A-purified GL-2045 by at least one of cation exchange chromatography, anion exchange flow-through, and hydrophobic interaction column chromatography. Polish In a preferred embodiment, it has been determined that a multi-step purification process is desirable, including purification of GL-2045 by Protein A and purification of Protein A-purified GL-2045 by all three of cation exchange chromatography in binding mode, anion exchange in flow-through mode, and a hydrophobic interaction column in either binding or flow-through mode. Polish A four-step purification process is preferred, including: (a) a purification step comprising: (i) a step of filtration of the GL-2045 composition with a depth of 100 μm; (ii) a step of filtration of the GL-2045 composition with a depth of 100 μm; (iii) a step of filtration of the GL-2045 composition with a depth of 100 μm; (iv) a step of filtration of the GL-2045 composition with a depth of 100 μm; (v) a step of filtration of the GL-2045 composition with a depth of 100 μm; (vi ...

[0062] upstream manufacturing Generally speaking, an upstream production method is a method in which biological material is inoculated and grown in culture under controlled conditions to produce a specific type of protein biological product (e.g., GL-2045). As used herein, "upstream production method" specifically refers to a method for recombinant production of a protein, without reference to subsequent purification and filtration steps that are generally classified as downstream production methods. Upstream production methods with modifications or changes aimed at optimizing the properties of a specific protein (e.g., multimerization efficiency) are referred to herein as "optimized upstream production methods." To produce a final protein product with specific properties, several aspects of upstream protein production can be optimized (e.g., changed or modified to achieve a desired result). Aspects of upstream recombinant protein production that can be optimized can include, but are not limited to, the composition of the expression vector encoding the protein, cell type, basal medium, media additives including feed, feeding schedule, passaging schedule, culture temperature, temperature shift, humidity, degree of aeration, pH, seeding cell density, CO2 level, and / or oxygen level. In some embodiments, the optimized upstream manufacturing methods described herein result in the production of high titers of GL-2045 with an increased percentage of higher order multimers compared to GL-2045 produced by non-optimized upstream manufacturing methods.

[0063] In some embodiments of the present invention, Chinese hamster ovary (CHO) cells are transfected with an expression vector encoding GL-2045. In some embodiments, insertion of the GL-2045 expression cassette into the genome is mediated by a piggyBac transposon, in which the GL-2045 expression cassette is flanked by piggyBac minimal inverted repeat elements. Coexpression of this GL-2045 expression vector with a vector encoding piggyBac transposase mediates gene integration into an actively transcribed region of the genome, resulting in the generation of cell lines with stable and enhanced gene expression compared to standard transfection methods (see US2010 / 0311116 and Matasci et al., Biotechnol. Bioeng. V. 108, pp. 2141-2140, (2011), incorporated herein by reference). The piggyBac system typically increases protein production, at least in part due to the large number of integrated transgenes. However, although the inventors selected a high-titer, high-viability clonal cell line with a relatively low transgene insertion rate (e.g., as determined by UV spectrophotometry and 11 copies), one of skill in the art would understand that the use of selective antimicrobial pressure would also allow for the use of transgene insertion rates of greater than about 50 inserted copies or greater than about 100 inserted copies. In some embodiments, the expression vector comprises a nucleic acid encoding a leader peptide (e.g., SEQ ID NO: 1). In some embodiments, the expression vector further comprises an antimicrobial resistance gene to allow for the selection of successfully transfected CHO cells. In certain embodiments of the invention, successfully transfected CHO cells are generated in the absence of antimicrobial selection (see US2010 / 0311116). In some embodiments, the expression vector further comprises a transcription enhancer (e.g., a CMV enhancer) to promote high-level expression of GL-2045.

[0064] In some embodiments, CHO cells transfected with a GL-2045 expression vector are cultured in a bioreactor. In some embodiments, a CHO cell line with many mutations is carefully selected for a single stable transfection with the GL-2045 expression vector using a technique that preferentially induces insertion at transcriptionally active sites. In some embodiments, the culture conditions applied to the selected transfected CHO cell line allow the culture protocol to continue longer than standard manufacturing methods without adversely affecting cell viability. Standard manufacturing methods can average 12 days in a bioreactor, at which point there is a decrease in cell viability due to the increase in cell debris present in the culture. In addition to being associated with a loss of cell viability, the cell debris dramatically increases the challenges associated with filtration and purification. In some embodiments of the invention, cells are seeded into a bioreactor at a predetermined cell density and cultured for >12 days. In some embodiments, the cells are cultured in a bioreactor with an acceptable viable cell density for 13, 14, 15, 16, 17, 18, 19, 20, 21 days or more.

[0065] In some embodiments, ActiCHO P is used as the basal medium for optimized upstream production of optimally produced GL-2045. The terms "ActiCHO P" and "optimized medium," as used interchangeably herein, refer to the commercially available ActiCHO® basal medium ("ActiCHO P," GE Healthcare), any substantial copy thereof, or a medium containing substantially the same constituents in substantially the same amounts as ActiCHO P. ActiCHO P is also currently marketed by GE as Hyclone "ActiPro," which is nearly the same product as ActiCHO P and is an equivalent reagent for purposes of these disclosures. In some embodiments, ActiCHO® Feed A and Feed B (also currently marketed by GE as Hyclone "Cell Boost 7a" and Hyclone "Cell Boost 7b," which are the same product as ActiCHO® Feed A and Feed B and are equivalent reagents for purposes of this disclosure) are used in addition to the basal medium. The terms "ActiCHO Diet A" or "Optimized Diet A," as used interchangeably herein, and "ActiCHO Diet B" or "Optimized Diet B," as used interchangeably herein, refer to the commercially available ActiCHO® feed (GE Healthcare), a substantial copy thereof, or a feed containing substantially the same constituents in substantially the same amounts as ActiCHO Diet A and / or ActiCHO Diet B. In some embodiments of the invention, CHO cells transfected with a GL-2045 expression vector are fed ActiCHO Diet A and Diet B daily. In some embodiments of the invention, CHO cells transfected with a GL-2045 expression vector are fed ActiCHO Diet A and Diet B every other day. In some embodiments of the invention, CHO cells transfected with a GL-2045 expression vector are fed ActiCHO Diet A and Diet B via continuous feeding.

[0066] In some embodiments of the optimized upstream manufacturing method, CHO cells transfected with a GL-2045 expression vector are grown to a particular cell density before the temperature shift. In some aspects, the CHO cells are grown to a density of about 5-30 million cells / mL before the temperature shift. In some aspects, the cells are grown to a density of about 6, 7, 8, 9, 10, 15, 20, 25, or about 30 million cells / mL before the temperature shift. In some aspects, the CHO cells are grown to a density of about 10-25 million cells / mL before the temperature shift. In some aspects of the optimized manufacturing method, the CHO cells are grown to a density of about 10-15 million cells / mL before the temperature shift.

[0067] In some embodiments, CHO cells transfected with a GL-2045 expression vector are cultured at 37°C ± 1°C until a predetermined cell density is reached. In some aspects, the temperature is shifted to 32.5°C ± 1°C after the cells reach a predetermined cell density. This aspect contrasts with previously described culture methods for recombinant protein production, in which cells are often cultured at 37°C for a predetermined number of days and then the temperature is shifted to 31°C (Ouguchi et al., Cytotechnology, 52(3), pp. 199-207, (2006); Masterson and Smales, Pharmaceutical Bioprocessing, 2(1), pp. 49-61, (2014)). The inventors have determined that a temperature shift from 37°C ± 1°C to 32.5°C ± 1°C based on cell density rather than culture time unexpectedly provides a combination of increased viability, improved cell density, and substantially increased protein titer relative to standard upstream manufacturing methods. In some embodiments, CHO cells transfected with a GL-2045 expression vector are subjected to a double temperature shift. In one embodiment, the transfected CHO cells are cultured at 37°C ± 1°C and shifted to 34°C ± 1°C before reaching peak viable cell density. In a preferred embodiment, this temperature shift occurs while the CHO cells are maintained in log growth phase. In a particularly preferred embodiment, the first temperature shift occurs on day 3 or 4 of culture. In another embodiment, the transfected CHO cells are cultured at 37°C ± 1°C until a predetermined cell density is reached, at which time the temperature is shifted to 34°C ± 1°C. In a preferred embodiment, the cell density is 5 to 20 million cells / ml at the first temperature shift. In a particularly preferred embodiment, the cell density is 8 to 15 million cells / ml at the first temperature shift. In some embodiments, a second temperature shift occurs on day 7 ± 1. In some embodiments, the temperature is then further shifted to 31°C. In some embodiments, this second temperature shift occurs about 4 days after the first temperature shift.

[0068] Downstream manufacturing method In some embodiments, harvesting GL-2045 is achieved by downstream production methods. In some embodiments, downstream production methods are used in combination with the optimized upstream production methods described herein to remove or isolate specific protein fractions (e.g., removal of disordered, high molecular weight aggregates of homodimeric GL-2045). As used herein, a "downstream production method" is a protein purification and filtration step performed on a protein supernatant to generate a protein composition of a desired purity and / or concentration. In some embodiments, the downstream production method is optimized for purification and filtration of GL-2045 to produce and / or maintain a specific multimerization profile of GL-2045, referred to herein as an "optimized downstream production method."

[0069] In some embodiments, GL-2045 is purified by affinity chromatography. In some embodiments, GL-2045 is purified using a Protein A column. As described above, Protein A columns are very expensive and are reused a significant number of times to be economically viable. Reusing a Protein A column necessarily involves "regeneration" of the Protein A column to maintain its protein binding capacity. As used herein, "regeneration" or "regenerating" refers to the removal of bound proteins from the Protein A column that were not removed during the elution process. In some embodiments, the Protein A column must be regenerated during GL-2045 purification more frequently than indicated in the manufacturer's instructions or more frequently than is conventional in the art for purifying monoclonal antibodies or Fc-fusion proteins. In some embodiments, the Protein A column must be regenerated at least twice as frequently as recommended by the manufacturer. In further embodiments, the Protein A column must be regenerated between each successful round of passage of GL-2045 supernatant over the column. In such embodiments, purification of GL-2045 using a Protein A affinity column entails the use of a high stringency regeneration buffer to remove avidly bound GL-2045 multimers from the Protein A column and regenerate the full binding capacity of the Protein A column. In preferred embodiments, a high stringency regeneration buffer is associated with no or little degradation of the Protein A column. In some embodiments, the high stringency regeneration buffer comprises a soluble base. In some embodiments, the base is sodium hydroxide (NaOH). In some embodiments, the regeneration buffer has an NaOH concentration greater than 0.3 M NaOH. For example, a high stringency regeneration buffer can be greater than 0.35 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.8 M, 0.9 M, 1.0 M NaOH, or more. In certain embodiments, the concentration of NaOH in the regeneration buffer is 0.5 M NaOH.

[0070] In some embodiments, elution of GL-2045 from a Protein A affinity column is optimized to remove or reduce the amount of high molecular weight, disordered aggregates of GL-2045 from the pharmaceutical substance. In some embodiments, GL-2045 is eluted from the Protein A column by an elution gradient (e.g., a pH elution gradient).

[0071] In some embodiments, the optimized downstream manufacturing process for GL-2045 comprises purification by affinity chromatography (e.g., Protein A affinity chromatography) and at least one or more methods selected from cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction columns. Polish In a preferred embodiment, the method includes a multi-step purification process, including purification by affinity chromatography (e.g., protein A affinity chromatography), and purification by each of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction column chromatography. Polish A four-step purification process for GL-2045 is used instead of the two or three step purification processes commonly practiced in the art, including: Polish The term "" classically refers to the post-protein A purification removal of remaining impurities, including aggregates, endotoxins, DNA, and / or viruses. Additionally, with respect to GL-2045, " Polish " also means controlling the proportion of homodimers and specific higher order multimers, such as through the use of these same chromatographic techniques.

[0072] In some embodiments, GL-2045 is purified by ion exchange chromatography (e.g., cation or anion exchange). Polish In some embodiments, GL-2045 is purified by ion exchange chromatography. PolishThe purification is performed using an elution buffer that reduces and / or minimizes the amount of disordered, high molecular weight aggregates of GL-2045 homodimers retained during post-Protein A purification processing. In some embodiments, step elution is performed to elute GL-2045 from the Protein A column. In some embodiments, gradient elution is performed to elute GL-2045 from the Protein A column. In some embodiments, the elution buffer is a sodium acetate buffer. In some embodiments, the concentration of sodium acetate in the elution buffer is at least 25 mM. For example, the concentration of sodium acetate in the elution buffer can be at least 30, 35, 40, 45, 50, 55, 60, 75, 100 mM, or more, sodium acetate. In some embodiments, the elution buffer is 50 mM sodium acetate. In some embodiments, the elution buffer is 50 mM sodium acetate with the addition of a variable amount of additional salt buffer (e.g., NaCl buffer). In some embodiments, the additional buffer is a 1 M NaCl, pH 5 buffer ("Buffer B"). In some embodiments, the elution buffer comprises at least 30% Buffer B. In some embodiments, the elution buffer comprises 30% to 40% Buffer B. In some embodiments, the elution buffer comprises 35% to 40% Buffer B. In still further embodiments, the elution buffer comprises 37% to 39% Buffer B. In some embodiments, the elution buffer comprises 38% + / - 0.5% Buffer B.

[0073] In some embodiments, GL-2045 is purified using hydrophobic interaction chromatography (HIC). PolishIn some embodiments, an HIC column (e.g., an Octyl FF HIC column) is selected to remove high molecular weight disordered aggregates of GL-2045. Purification of GL-2045 using an HIC column can be performed in either flow-through or binding mode. Those skilled in the art will appreciate that adjusting variables such as pH and salt conditions will determine whether GL-2045 binds to the HIC resin or flows through the column. In some embodiments, homodimers and / or higher multimers (e.g., butyl HP and / or phenyl HP) are removed. An HIC column is selected to purify a specific fraction of GL-2045, such as a GL-2045-specific fraction (e.g., a GL-2045-specific fraction) for the treatment of a specific disease indication. In some embodiments, it may be desirable to isolate a specific fraction of GL-2045 for the treatment of a specific disease indication. For example, an HIC column may be used to generate a pharmaceutical agent composed of a specific GL-2045 fraction (e.g., a pharmaceutical agent composed primarily of GL-2045 homodimers, a pharmaceutical agent composed primarily of dimers of homodimers, a pharmaceutical agent composed of higher-order multimers of GL-2045, etc.). Separation of GL-2045 fractions into distinct products may be beneficial for specific disease indications. For example, GL-2045 homodimers bind to FcγRI but substantially bind to other FcRs. Thus, GL-2045 homodimers may be particularly useful for treating diseases mediated, at least in part, by FcγRI signaling, such as peritonitis (Heller et al., J. Immunol., Vol. 162, 1992) or acute lung injury (Xie et al., J. Immunol., Vol. 188, 2012). Similarly, trimers, and potentially dimers and tetramers, of GL-2045 may be particularly useful for treating autoimmune diseases (see WO 2015 / 168643). Because C1q is a hexamer, pentamers, hexamers, and heptamers may be particularly useful for treating complement-mediated diseases. Those skilled in the art will recognize that these are just a few of the ways in which GL-2045 fractions may be beneficial for treating specific diseases.

[0074] In some embodiments, the optimized downstream manufacturing methods described herein can be a combination of individual purification and / or filtration techniques. For example, in some embodiments, the optimized downstream method involves purifying GL-2045 by affinity chromatography (e.g., by an optimized method on a Protein A column), followed by additional purification by an ion exchange chromatography method. Polish (e.g., by optimized cation exchange methods) Polish ), and / or hydrophobic interaction columns. In some embodiments, the optimized downstream methods described herein include a four-step purification process, including purification by Protein A, cation exchange, anion exchange flow-through, and hydrophobic interaction column. In some embodiments, additional depth filtration and / or ultrafiltration steps may also be used.

[0075] Thus, the terms "optimized production method" or "optimized production method," as used interchangeably herein, can refer to optimized upstream and / or optimized downstream production methods. In some embodiments, optimized production methods include both optimized upstream and downstream methods. Thus, as used herein, the terms "optimally produced stradomer" or "optimally produced GL-2045" refer to a high-titer, high-order multimer-predominant GL-2045 composition produced according to optimized upstream production conditions and / or optimized downstream production methods. While the GL-2045 compositions described herein can be optimally produced GL-2045 (i.e., GL-2045 produced by the methods described herein), one of skill in the art will understand that GL-2045 compositions falling within the defined multimer patterns described herein can be achieved by other means. Thus, "GL-2045 composition" or "recombinant GL-2045 composition" or "purified GL-2045 composition" refers to a composition containing GL-2045, including the GL-2045 drug substance, regardless of whether the composition has been produced via an optimal manufacturing method. As used herein, the terms "multimer pattern" or "banding pattern" or any similar term are used interchangeably and refer to the pattern of multimers observed in an analytical assay of a GL-2045 composition. An exemplary multimer pattern is shown in Figure 33.

[0076] In some embodiments, provided herein are recombinantly produced GL-2045 compositions with defined multimer patterns. As used herein, the terms "defined multimer pattern" or "defined multimerization pattern" or "defined banding pattern" refer to a pattern of GL-2045 multimerization that is reproducible and can be described in terms of the percentage of the overall GL-2045 composition that exists as homodimers, higher order multimers, and / or highest order multimers. Those skilled in the art will understand that the absolute percentage of homodimers and / or multimers may vary based on the analytical method used. For example, digital software analysis of an SDS-PAGE gel will yield somewhat different multimer percentages compared to analytical SEC-HPLC of the same composition. Unless otherwise specified herein, the percentages of homodimers and multimers of the GL-2045 compositions described herein are expressed as percentages measured by analytical SEC-HPLC methods. In some embodiments, recombinantly produced GL-2045 is provided, wherein at least 80% of the GL-2045 exists as a non-homodimer or "multimer" (e.g., dimers of homodimers, trimers of homodimers, etc.). In some embodiments, greater than 80% of the GL-2045 exists as a multimer. For example, optimized production methods such as those described herein may result in GL-2045 protein compositions in which 80%, 85%, 90%, 95% or more of the GL-2045 exists as a multimer. In some embodiments, at least 30% of the GL-2045 exists as a "highest order multimer," defined herein as a homodimeric 7-mer or greater. In some embodiments, no more than 40% of the recombinantly produced GL-2045 exists as a highest order multimer. One of skill in the art will understand that when speaking of "bands" or "fractions," the number of bands encompasses the number of homodimers present in a fraction, unless otherwise specified. Thus, for example, a 2 band contains a dimer of a homodimer, and a 3 band contains a trimer of a homodimer.Thus, for example, 2 bands contain a dimer of a homodimer, 3 bands contain a trimer of a homodimer, 4 bands contain a tetramer of a homodimer, and so on.

[0077] As used herein, "higher order multimers" refers to trimers or higher of homodimers (i.e., multimers present in 3 or more fractions). As used herein, the term "highest order multimer" refers to multimers in 7 or more fractions, or fractions containing 7 or more homodimer 7-mers.

[0078] In some embodiments, recombinantly produced GL-2045 compositions are provided, wherein the homodimer fraction comprises less than about 20% of the total composition. In some embodiments, the homodimer fraction comprises about 12% to about 19%, about 14% to about 19%, about 15.5% to about 17.5%, or about 14% to about 18.5% of the total protein composition. In some embodiments, the homodimer fraction comprises about 15.9% or about 16.2% of the total protein composition.

[0079] In some embodiments, recombinantly produced GL-2045 compositions are provided in which the dimer fraction of homodimers constitutes about 7% to about 13%, about 7% to about 12.5%, about 7% to about 12%, about 9% to about 11%, or about 9.1% to about 11.7% of the total composition. In some embodiments, the dimer fraction of homodimers constitutes about 10% or about 10.6% of the total protein composition.

[0080] In some embodiments, recombinantly produced GL-2045 compositions are provided in which the homodimer trimer fraction comprises about 5.5% to about 11%, about 5.5% to about 10%, or about 6.5% to about 8% of the total composition. In some embodiments, the homodimer trimer fraction comprises about 7% or about 7.3% of the total protein composition.

[0081] In some embodiments, recombinantly produced GL-2045 compositions are provided in which the homodimeric tetramer fraction comprises about 10% to about 16%, about 11% to about 16%, about 13% to about 15%, or about 12.4% to about 15.1% of the total composition. In some embodiments, the homodimeric tetramer fraction comprises about 14% or about 14.3% of the total protein composition.

[0082] In some embodiments, recombinantly produced GL-2045 compositions are provided in which the pentameric fraction of homodimers constitutes about 6% to about 9%, about 7% to about 8%, or about 7.1% to about 8.2% of the total composition. In some embodiments, the dimers of the pentameric fraction constitute about 7% or about 7.5% of the total protein composition.

[0083] In some embodiments, recombinantly produced GL-2045 compositions are provided in which the homodimeric hexamer fraction comprises about 10% to about 14%, about 12% to about 13%, or about 12.1% to about 13.2% of the total composition, hi some embodiments, the homodimeric hexamer fraction comprises about 12.7% or about 12.6% of the total protein composition.

[0084] In some embodiments, recombinantly produced GL-2045 compositions are provided, wherein the highest order multimer fraction comprises at least about 28% of the total composition. In some embodiments, the highest order multimer fraction comprises no more than about 35% of the total protein composition. In some embodiments, the highest order multimer fraction comprises about 30% to about 34%, or about 28.6% to about 35.1% of the total protein composition. In some embodiments, the highest order multimer fraction comprises about 31.4% or about 31.9% of the total protein composition. In some embodiments, the homodimer fraction comprises less than about 20% of the total composition, the highest order multimer fraction comprises at least about 28% of the total composition, the dimer fraction of homodimers comprises about 7% to about 13% of the total composition, the trimer fraction of homodimers comprises about 5% to about 11% of the total composition, the tetramer fraction of homodimers comprises about 10% to about 16% of the total composition, the pentamer fraction of homodimers comprises about 6% to about 10% of the total composition, and the hexamer fraction of homodimers comprises about 10% to about 14% of the total fraction. % of the total composition, the homodimer dimer to homodimer hexamer fractions comprising about 40% to about 60% of the total composition, the homodimer trimer to homodimer hexamer fractions comprising about 32% to about 50% of the total composition, the homodimer tetramer to homodimer hexamer fractions comprising about 30% to about 37% of the total composition, the homodimer pentamer to homodimer hexamer fractions comprising about 18% to about 23% of the total composition, or any combination of the foregoing.

[0085] In some embodiments, a recombinantly produced GL-2045 composition is provided, wherein approximately 80% of the total composition comprises homodimers or more (i.e., two or more bands). In some embodiments, approximately 60-80%, 62-80%, or 60-78% of the total composition comprises homodimers or more (i.e., band 3 or more). In some embodiments, about 54-76%, about 54-72%, about 56-76%, or about 54-67% of the total composition comprises homodimers or more (i.e., band 4 or more). In some embodiments, a GL-2045 composition is provided, wherein approximately 44-60%, 44-57%, or 44-51% of the total composition comprises pentamers or more (i.e., band 5 or more). In some embodiments, a GL-2045 composition is provided, wherein approximately 38-51% of the total composition comprises hexamers or greater (i.e., band 6 or greater).

[0086] In some embodiments, recombinantly produced GL-2045 is provided, wherein 2 to 6 bands (i.e., homodimer dimers to homodimer hexamers) comprise about 39 to 61% or about 44 to 60% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, wherein 3 to 6 bands (i.e., homodimer trimers to homodimer hexamers) comprise about 32 to 50% or about 35 to 48% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, wherein 4 to 6 bands (i.e., homodimer tetramers to homodimer hexamers) comprise about 26 to 39% or about 30 to 39% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, wherein 5 to 6 bands (i.e., homodimer pentamers to homodimer hexamers) comprise about 16 to 23% or about 18 to 23% of the composition.

[0087] Without being bound by theory, at least the active components of GL-2045 in binding to low-affinity Fc receptors and C1q are homodimers and dimers of homodimers. Those skilled in the art will readily understand that the amount of homodimers or homodimers and dimers in the final product can be reduced using the optimized chromatography methods described herein or similar purification techniques. Therefore, those skilled in the art will know that doing so will change the percentage of multimers described herein. By way of example and without limiting the generality of the foregoing, if those skilled in the art remove 50% of homodimers or homodimers and dimers in the purification process, the percentage of each remaining multimer (i.e., trimers, tetramers, pentamers, hexamers, 7-mers, etc.) will increase accordingly. Removal of 90% of the homodimers and 50% of the dimers will reduce the total protein present in the final product by approximately 20% + / - 5%, and therefore increase the percentage of trimers, tetramers, pentamers, hexamers, and 7-mers expressed as a percentage of total protein.

[0088] Moreover, those skilled in the art will further recognize that current chromatographic techniques generally do not allow for the removal or reduction of a single multimer band, such as a highest-order multimer, without simultaneously removing adjacent bands, such as hexamers to some extent, and pentamers to a lesser extent. Thus, those skilled in the art will know that the observed compensatory increase in the percentage of any given homodimer as a result of removing or reducing the highest-order multimer will increase to a greater extent than the given multimer from the fraction of GL-2045 that is removed (e.g., the percentage of homodimers will increase to a greater extent than the percentage increase observed for hexamers when the highest-order multimer is removed or reduced). In either case, the cumulative increase in the multimer percentage of the remaining multimers should be equal to the proportion of multimers for the removed fraction, depending on any variability due to the analytical method.

[0089] Pharmaceutical Composition Administration of the GL-2045 compositions described herein may be via any common route, orally, parenterally, or topically. Exemplary routes include, but are not limited to, oral, nasal, buccal, rectal, vaginal, ocular, subcutaneous, intramuscular, intraperitoneal, intravenous, intraarterial, intratumoral, spinal, intrathecal, intraarticular, intraarticular, subarachnoid, sublingual, oral mucosal, bronchial, lymphatic, intrauterine, subcutaneous, intratumoral, on an implantable device such as a suture, or integrated into an implantable device such as an implantable polymer, intradural, intracortical, or dermal. Such compositions will typically be administered as pharmaceutically acceptable compositions as described herein. In a preferred embodiment, an isolated, optimally engineered stradomer is administered intravenously or subcutaneously.

[0090] As used herein, the term "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

[0091] The GL-2045 compositions of the present invention may be formulated in a neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with the free amino groups of proteins) formed with inorganic acids such as hydrochloric acid or phosphoric acid, or organic acids such as acetic acid, oxalic acid, tartaric acid, mandelic acid, etc. Salts formed with free carboxyl groups can also be derived from inorganic bases such as sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

[0092] Sterile injectable solutions are prepared by incorporating GL-2045, optimally prepared, in the required amount in a suitable solvent containing various other ingredients listed above, followed by filter sterilization, as needed. In some embodiments, sterile injectable solutions are formulated for intramuscular, subcutaneous, or intravenous administration. Generally, dispersions are prepared by incorporating various sterilized active ingredients into a sterile vehicle containing the basic dispersion medium and the required other ingredients listed above. In the case of sterile powders for preparing sterile injectable solutions, the preferred preparation method is vacuum drying and freeze-drying, which yields a powder of the active ingredient plus any additional desired ingredients from its previously sterile-filtered solution.

[0093] Additionally, one embodiment is a GL-2045 composition suitable for oral administration, provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier should be assimilable or edible, and include liquid, semi-solid (e.g., paste), or solid carriers. Except insofar as any conventional medium, agent, diluent, or carrier is deleterious to the acceptability or therapeutic efficacy of the optimally formulated stradomer formulation contained therein, its use in an orally administrable optimally formulated stradomer composition for use in the practice of the methods of the present invention is suitable. Examples of carriers or diluents include fats, oils, water, saline, lipids, liposomes, resins, binders, fillers, etc., or combinations thereof. As used herein, the term "oral administration" includes oral, buccal, enteral, or gastric administration.

[0094] In one embodiment, the GL-2045 composition is combined with the carrier in any conventional and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, microencapsulation, absorption, etc. Such procedures are routine to those skilled in the art.

[0095] In certain embodiments, a GL-2045 composition in powder form is thoroughly combined or mixed with a semi-solid or solid carrier. Mixing can be performed by any conventional method, such as milling. Stabilizers can also be added during the mixing process to protect the composition from loss of therapeutic activity (e.g., through stomach denaturation). Examples of stabilizers for use in orally administrable compositions include buffers, antagonists to gastric acid secretions, amino acids such as glycine and lysine, carbohydrates such as glucose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, and mannitol, protease inhibitors, and the like. More preferably, for orally administered compositions, the stabilizer can also include an antagonist to gastric acid secretions.

[0096] Furthermore, the GL-2045 composition for oral administration combined with a semi-solid or solid carrier can be further formulated into a hard or soft shell gelatin capsule, tablet, or pill. More preferably, the gelatin capsule, tablet, or pill is enterically coated. The enteric coating prevents the composition from denaturing in the stomach or upper intestine, where the pH is acidic. See U.S. Patent No. 5,629,001. Upon reaching the small intestine, the basal pH inside dissolves the coating, releasing the composition to interact with intestinal cells, such as Peyer's patch M cells.

[0097] In another embodiment, the GL-2045 composition in powder form is thoroughly combined or mixed with a material to create nanoparticles that encapsulate the immunologically active biomimetic or to which the immunologically active biomimetic is attached. Each nanoparticle will have a size of 100 microns or less. The nanoparticles may have mucoadhesive properties that allow gastrointestinal absorption of immunologically active biomimetic that would otherwise not be orally bioavailable.

[0098] In another embodiment, a powdered composition is combined with a liquid carrier, such as water or saline, with or without a stabilizer.

[0099] A specific GL-2045 formulation that can be used is a solution of an immunologically active biomimetic protein in a hypotonic phosphate-based buffer containing 6 mM sodium phosphate monobasic monohydrate, 9 mM sodium phosphate dibasic heptahydrate, and 50 mM sodium chloride, potassium-free, at a pH of 7.0 + / - 0.1. The concentration of the immunologically active biomimetic protein in the hypotonic buffer can range from 10 μg / mL to 100 mg / mL. This formulation can be administered via any route of administration, including, but not limited to, intravenous administration.

[0100] Furthermore, the GL-2045 composition for topical administration combined with a semi-solid carrier can be further formulated into a cream or gel cartilage. A preferred carrier for forming gel cartilage is a gel polymer. Preferred polymers used to prepare the gel composition of the present invention include, but are not limited to, carbopol, carboxymethylcellulose, and pluronic polymers. Specifically, a powdered Fc multimer composition is combined with an aqueous gel containing a polymerizing agent such as Carbopol 980 at a strength of 0.5% wt% to 5% wt% / vol for application to the skin to treat diseases on or beneath the skin. As used herein, the term "topical administration" includes application to dermal, epidermal, subcutaneous, or mucosal surfaces.

[0101] Furthermore, GL-2045 compositions can be formulated into polymers for subcutaneous or subcutaneous implantation.Preferred formulations for implantable drug-injecting polymers are generally considered safe drugs, and may include, for example, cross-linked dextran (Samantha Hart, Master of Science Thesis, "Elution of Antibiotics from a Novel Cross-Linked Dextran Gel: Quantification" Virginia Polytechnic Institute and State University, June 8, 2009), dextran-tyramine (Jin, et al. (2010) Tissue Eng. Part A. 16 (8): 2429-40), dextran-polyethylene glycol (Jukes, et al. (2010) Tissue Eng. Part A., 16 (2): 565-73), or dextran-gluteraldehyde (Brondsted, et al. (1998) J. Controlled Release, 53: 7-13). Those skilled in the art will know that many similar polymers and hydrogels can be formed that incorporate stradomers anchored within the polymer or hydrogel and control the pore size to a desired diameter.

[0102] The solution is administered to the formulation in a manner compatible with the dosage formulation and in an amount that is therapeutically effective to improve or repair symptoms.The formulation is easily administered in a variety of dosage forms, such as ingestible solutions, drug-release capsules, etc.Some variations in administration may occur depending on the condition of the subject being treated.The person responsible for administration can always determine the appropriate dose for each individual subject.In addition, for human administration, the formulation meets the sterility, general safety, and purity standards as required by the FDA Center for Biologics Evaluation and Research standards.

[0103] The route of administration will necessarily vary with the location and nature of the condition being treated and may include, for example, intradermal, transdermal, subcutaneous, parenteral, intranasal, intravenous, intramuscular, intranasal, subcutaneous, transdermal, intratracheal, intraperitoneal, intratumor, perfusion, lavage, direct injection, rectal, and oral administration.

[0104] In one embodiment, the GL-2045 composition is administered intravenously, subcutaneously, orally, intraperitoneally, sublingually, buccally, transdermally, rectally, by subcutaneous implant, or intramuscularly. In a specific embodiment, the optimally manufactured stradomer is administered intravenously, subcutaneously, or intramuscularly. In one embodiment, the optimally manufactured stradomer is administered at a dose of about 0.005 mg / kg to about 1000 mg / kg. In a further embodiment, the optimally manufactured stradomer is administered at about 0.01 mg / kg to about 100 mg / kg. In yet a further embodiment, the optimally manufactured stradomer is administered at about 0.1 mg / kg to about 20 mg / kg. In yet a further embodiment, the optimally manufactured stradomer is administered at about 1 mg / kg to about 10 mg / kg. In yet a further embodiment, the optimally manufactured stradomer is administered at about 2 mg / kg to about 5 mg / kg. Optimally manufactured stradomers may be administered at least daily, weekly, biweekly, monthly, or sometimes at longer intervals. A biphasic dosing regimen may be used, with the first dosing phase comprising from about 0.1% to about 300% of the second dosing phase.

[0105] In further embodiments, the GL-2045 composition is administered before, during, or after the administration of one or more additional medical and / or therapeutic agents. In further embodiments, the additional pharmaceutically active agent comprises a steroid; a biologic anti-autoimmune drug such as a monoclonal antibody, fusion protein, or anticytokine; a non-biologic anti-autoimmune drug; an immunosuppressant; an antibacterial; and an antiviral agent; a cytokine; or an agent that can otherwise act as an immunomodulator. In still further embodiments, the steroid is prednisone, prednisolone, cortisone, dexamethasone, mometasone, testosterone, estrogen, oxandrolone, fluticasone, budesonide, beclomethasone, albuterol, or levalbuterol. In still further embodiments, the monoclonal antibody is selected from the group consisting of eculizumab, ocrelizumab, infliximab, adalimumab, rituximab, tocilizumab, golimumab, ofatumumab, LY2127399, belimumab, veltuzumab, mepolizumab, necitumumab, nivolumab, dinutuximab, secukinumab, evolocumab, blinatumomab, pembrolizumab, ramucirumab, vedolizumab, siltuximab, obinutuzumab, adotrastuzumab, raxibacumab, In yet a further embodiment, the fusion protein is etanercept or abatacept. In yet a further embodiment, the anti-cytokine biologic is anakinra. In still further embodiments, the anti-rheumatic non-biologic drug is cyclophosphamide, methotrexate, azathioprine, hydroxychloroquine, leflunomide, minocycline, an organogold compound, fostamatinib, tofacitinib, etoricoxib, or sulfasalazine.In still further embodiments, the immunosuppressant is cyclosporine A, tacrolimus, sirolimus, mycophenolate mofetil, everolimus, OKT3, antithymocyte globulin, basiliximab, daclizumab, or alemtuzumab. In still further embodiments, the optimally manufactured stradomer is administered before, during, or after administration of a chemotherapeutic agent. In still further embodiments, the optimally manufactured stradomer and the additional therapeutic agent exhibit therapeutic synergy when administered together. In one embodiment, the optimally manufactured stradomer is administered before administration of the additional therapeutic agent. In another embodiment, the optimally manufactured stradomer is administered simultaneously with administration of the additional therapeutic agent. In yet another embodiment, the optimally manufactured stradomer is administered with and after administration of the additional therapeutic agent.

[0106] In one embodiment, the GL-2045 composition is administered covalently affixed to an implantable device. In one embodiment, the optimally manufactured stradomer is affixed to a suture. In another embodiment, the optimally manufactured stradomer is affixed to a graft or stent. In another embodiment, the optimally manufactured stradomer is affixed to a heart valve, orthopedic joint replacement, or implanted electronic lead. In another embodiment, the optimally manufactured stradomer is affixed to and embedded within an implantable matrix. In a preferred embodiment, the optimally manufactured stradomer is affixed to and embedded within an implantable hydrogel. In one embodiment, the hydrogel is composed of dextran, polyvinyl alcohol, sodium polyacrylate, or an acrylate polymer. In a further embodiment, the optimally manufactured stradomer is administered affixed within a hydrogel with pores large enough to allow entry of immune cells to interact with the affixed stradomer and then return to the blood circulation. In a further embodiment, the pore size of the hydrogel is between 5 and 50 microns. In a preferred embodiment, the pore size of the hydrogel is between 25 and 30 microns.

[0107] In another embodiment, the GL-2045 composition is administered to treat humans, non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, cows, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish, and reptiles bearing species-specific or chimeric stradomer molecules. In another embodiment, the human is an adult or a child. In yet another embodiment, the optimally manufactured stradomers are administered to prevent complement-mediated diseases. In a further embodiment, the stradomers are administered to prevent vaccine-associated autoimmune conditions in companion animals and livestock.

[0108] As used herein, the term "parenteral administration" includes any form of administration in which a compound is absorbed into a subject without absorption through the intestine. Exemplary parenteral administrations for use in the present invention include, but are not limited to, subcutaneous, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, intranasal, or intraarticular administration.

[0109] In addition, the GL-2045 compositions of the present invention may optionally be administered before, during, or after another pharmaceutical agent.

[0110] Below are specific examples of classifications of various pharmaceutical formulations and preferred routes of administration for specific exemplary diseases, as indicated:

[0111] Orally or sublingually dissolvable tablets: Angina, polyarteritis nodosa.

[0112] Intravenous, intramuscular, or subcutaneous: Myasthenia gravis, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), membranous nephropathy, neuromyelitis optica, antibody-mediated rejection of allografts, lupus nephritis, membranoproliferative glomerulonephritis (MPGN), idiopathic thrombocytopenic purpura, inclusion body myositis, paraproteinemic IgM demyelinating polymyalgia Neuropathy, gangrenous fasciitis, pemphigus, gangrene, dermatomyositis, granuloma, lymphoma, pneumonitis, aplastic anemia, multisystem organ failure, multiple myeloma, monoclonal gammopathy of undetermined significance, chronic inflammatory demyelinating polyneuropathy, inflammatory myopathy, thrombotic thrombocytopenic purpura, myositis, anemia, neoplasia, hemolytic anemia, encephalitis, myelitis, human T-cell leukemia virus Myelopathy, leukemia, multiple sclerosis and optic neuritis, particularly associated with type 1 HCV, asthma, epidermal necrolysis, Lambert-Eaton myasthenic syndrome, neuropathy, uveitis, Guillain-Barré syndrome, graft-versus-host disease, stiff-man syndrome, paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelopathy and sensory neuropathy with anti-Hu antibodies, systemic vasculitis, systemic lupus erythematosus, autoimmune diabetic neuropathy, acute idiopathic autonomic neuropathy, Vogt-Koyanagi-Harada syndrome, multifocal motor neuropathy, lower motor neuron syndrome associated with anti- / GM1, demyelination, membranoproliferative glomerulonephritis, cardiomyopathy, Kawasaki disease, rheumatoid arthritis, and Evans syndrome, CIDP, MS, dermatomyositis, and muscular dystrophy. As used herein, the term "intravenous administration" includes all techniques for delivery to the systemic circulation via intravenous infusion or infusion.

[0113] Skin gels, lotions, creams, or patches: Vitiligo, shingles, acne, cheilitis.

[0114] Rectal suppositories, gels, or drops: Ulcerative colitis, hemorrhoidal inflammation.

[0115] Orally as a pill, lozenge, encapsulated, or enteric coated: Crohn's disease, celiac disease, irritable bowel syndrome, inflammatory liver disease, Barrett's esophagus.

[0116] Intracortical: epilepsy, Alzheimer's disease, multiple sclerosis, Parkinson's disease, Huntington's disease.

[0117] Intraperitoneal instillation or implantation: endometriosis.

[0118] Medical devices: coronary stents, coated on artificial joints.

[0119] Therapeutic Uses of Optimally Manufactured GL-2045 In one embodiment, a method is provided for treating or preventing a disease or condition, such as an autoimmune disease, an inflammatory disease, or a complement-mediated disease or condition.

[0120] Based on rational design and in vitro and in vivo validation, the optimally manufactured GL-2045 of the present invention will serve as an important biopharmaceutical for treating inflammatory diseases and disorders, as well as for modifying immune function in a variety of other settings, such as bioimmunotherapy for allergies, cancer, autoimmune diseases, infectious diseases, and inflammatory diseases. Medical conditions suitable for treatment with the immunologically active, optimally manufactured GL-2045 disclosed herein include any disease caused by or associated with complement activation or complement-mediated effector function, including increased or inappropriate complement activity. Such medical conditions include those currently or previously treated with complement-binding drugs, such as eculizumab. Eculizumab binds to complement protein C5 (a complement protein downstream of C1 and C1q in the classical complement pathway) and inhibits its cleavage and subsequent complement-mediated cell lysis. The biomimetic of the present invention provides a safe and effective alternative to other complement-binding drugs known in the art. For example, in some embodiments, the biomimetics of the invention bind C1q, the first subunit in the C1 complex of the classical complement pathway. Medical conditions suitable for treatment with immunologically active, optimally engineered stradomers include, but are not limited to, myasthenia gravis, hemolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal hemoglobinuria (PNH), membranous nephropathy, neuromyelitis optica, antibody-mediated rejection of allografts, lupus nephritis, macular degeneration, sickle cell disease, and membranoproliferative glomerulonephritis (MPGN).Additional medical conditions suitable for treatment with the immunologically active, optimally manufactured GL-2045 described herein are those currently routinely treated with a wide range of immunosuppressive therapies, such as autoimmune cytopenias, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, myasthenia gravis, anti-factor VIII autoimmune diseases, dermatomyositis, vasculitis, and uveitis, for which hIVIG has been found clinically useful (FG van der Meche et al., N. Engl. J. Med. 326, 1123 (1992); P. Gajdos et al., Lancet i, 406 (1984); Y. Sultan, M. et al., Lancet ii, 765 (1984); MCDalakas et al., N. Engl. J. Med. 329, 1993 (1993); D.R. Jayne et al., Lancet 337, 1137 (1991); P. LeHoang, et al., Ocul. Immunol. Inflamm. 8, 49 (2000)), as well as those cancer or inflammatory disease states for which monoclonal antibodies can be used or are already in clinical use. Conditions that can be effectively treated by the compounds of this invention include inflammatory diseases with an imbalance in the cytokine network, autoimmune diseases mediated by pathogenic autoantibodies or autoaggressive T cells, or acute or chronic relapsing autoimmune, inflammatory, or infectious diseases or processes.

[0121] In addition, other medical conditions that have an inflammatory component involving complement would benefit from treatment with GL-2045 compositions, such as asthma, lupus erythematosus, glomerulonephritis, glomerular nephropathy, arthritis, autoantibody-mediated diseases including autoimmune hemolytic anemia and autoimmune heart disease, multiple sclerosis, amyotrophic lateral sclerosis, Huntington's disease, Alzheimer's disease, Parkinson's disease, inflammatory bowel disease, paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, ischemia-reperfusion injury including, for example, myocardial infarction, spinal cord injury, and stroke, rejection of transplanted organs or blood, hepatitis B, hepatitis C, human immunodeficiency virus-associated inflammation, adrenoleukodystrophy, and epileptic disorders, particularly those thought to be associated with post-viral encephalitis including Rasmussen syndrome, West syndrome, and Lennox-Gastaut syndrome.

[0122] The general approach to treatment using the GL-2045 compositions described herein is to administer to a subject having a disease or condition a therapeutically effective amount of the GL-2045 composition to affect treatment. In some embodiments, the disease or condition can be broadly classified as an inflammatory disease with an imbalance in the cytokine network, an autoimmune disorder mediated by pathogenic autoantibodies or autoaggressive T cells, or a chronic relapsing disease or process of acute or chronic phase.

[0123] As used herein, the terms "treat" and "treatment" refer to administering a therapeutically effective amount of an optimally engineered stradomer of the present invention to a subject such that the subject has an improvement in the disease or condition, or symptoms of the disease or condition. An improvement is any amelioration or remediation of the disease or condition, or symptoms of the disease or condition. An improvement can be an observable or measurable improvement, or an improvement in the subject's general feeling of well-being. Thus, one of skill in the art will understand that treatment may improve the disease state, but may not be a complete cure for the disease. Specifically, improvement in a subject may include one or more of: reduced inflammation; reduced inflammatory laboratory markers such as C-reactive protein; reduced autoimmunity as evidenced by one or more of: autoimmune markers such as autoantibodies or improved platelet, white blood cell, or red blood cell counts; reduced rash or purpura; reduced weakness, numbness, or tingling; increased glucose levels in patients with hyperglycemia; reduced joint pain, inflammation, swelling, or deterioration; reduced muscle cramps and diarrhea frequency and volume; reduced angina; reduced tissue inflammation; or reduced seizure frequency; reduced cancer tumor burden; increased time to tumor progression; reduced cancer pain; increased survival or improved quality of life; or slowed or improved progression of osteoporosis.

[0124] As used herein, the term "therapeutically effective amount" refers to an amount that results in improvement or repair of the symptoms of a disease or condition. Those skilled in the art will understand that the therapeutically effective amount of GL-2045 produced herein may vary depending on the final drug substance. Thus, for example, if it is necessary to remove all lower-order multimers, a reduced dose of the resulting higher-order multimers may be required. Thus, there may be more than one "therapeutically effective dose" of GL-2045.

[0125] As used herein, "prevention" can mean preventing disease symptoms altogether, delaying the onset of disease symptoms, or reducing the severity of subsequently developed disease symptoms.

[0126] As used herein, the term "subject" is taken to mean any mammalian subject to which the optimally manufactured stradomers herein are administered according to the methods described herein. In certain embodiments, the methods of the disclosure are used to treat human subjects. The methods of the disclosure may also be used to treat non-human primates (e.g., monkeys, baboons, and chimpanzees), mice, rats, cows, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, bats, birds (e.g., chickens, turkeys, and ducks), fish, and reptiles to generate species-specific or chimeric stradomer molecules.

[0127] Complement inhibition has been demonstrated to reduce antibody-mediated diseases (see Stegall et al., American Journal of Transplantation 2011 Nov;11(1):2405-2413 - Epub 2011 Sept 22). Optimally engineered stradomers of the present invention may also be used to treat diseases or conditions that are antibody-mediated. Autoantibodies mediate many known autoimmune diseases and likely play a role in numerous other autoimmune diseases.Recognized antibody-mediated diseases for which the optimally engineered stradomers of the present invention may be used include anti-glomerular basement membrane antibody-mediated nephritis, including Goodbacher's disease; anti-donor antibodies (donor-specific alloantibodies) in solid organ transplantation; anti-aquaporin 4 antibodies in neuromyelitis optica; anti-VGKC antibodies in neuromyotonia, limbic encephalitis, and Marfan syndrome; anti-nicotinic acetylcholine receptor and anti-MuSK antibodies in myasthenia gravis; anti-VGCC antibodies in Lambert-Eaton myasthenic syndrome; anti-AMPAR and anti-GABA(B)R antibodies in limbic encephalitis, which is often associated with tumors; anti-GlyR antibodies in stiff-person syndrome or hyperalgesia; antiphospholipid, anticardiolipin, and anti-β2-glycoprotein I antibodies in recurrent spontaneous abortion, Hughes syndrome, and systemic lupus erythematosus; and anti-glutamic acid decarboxylation antibodies in stiff-person syndrome, autoimmune cerebellar ataxia, or limbic encephalitis. Acid enzyme antibodies; anti-NMDA receptor antibodies in a newly described syndrome involving both limbic and subcortical features with prominent movement disorders common in young adults and children, often associated with ovarian teratomas but which may be non-paraneoplastic; anti-double-stranded DNA, anti-single-stranded DNA, anti-RNA, anti-SM, and anti-C1q antibodies in systemic lupus erythematosus; antinuclear and antinucleolar antibodies in connective tissue diseases including scleroderma, Sjögren's syndrome, and polymyositis, including anti-Ro, anti-La, anti-Scl70, and anti-Jo-1; anti-rheumatoid factor antibodies in rheumatoid arthritis; anti-hepatitis B surface antigen antibodies in polyarteritis nodosa; anti-centromere antibodies in CREST syndrome; anti-streptococcal antibodies in endocarditis or as a risk for endocarditis; anti-thyroglobulin, anti-thyroid peroxidase, and anti-TSH receptor antibodies in Hashimoto's thyroiditis; and anti-U1 in mixed connective tissue disease and systemic lupus erythematosus. RNP antibodies; and anti-desmoglein and anti-keratinocyte antibodies in pemphigus.

[0128] The GL-2045 compositions of the present invention are useful in treating congestive heart failure (CHF), vasculitis, rosacea, acne, eczema, myocarditis and other conditions of the myocardium, systemic lupus erythematosus, diabetes, spondylosis, cerebrospinal fluid fibroblasts, and bone marrow stroma; osteoporosis; Paget's disease, giant cell tumor of bone; multiple myeloma; breast cancer; disuse osteoporosis; malnutrition, periodontal disease, Gaucher's disease, Langerhans cell granulomatosis, spinal cord injury, acute pneumococcal arthritis, osteomalacia, Cushing's syndrome, monostotic fibrous dysplasia, multiple fibrous dysplasia, periodontal remodeling. , and fractures; sarcoidosis; osteolytic bone cancer, lung cancer, kidney cancer, and rectal cancer; bone metastases, bone pain management, and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathies; transplant rejection, viral infections, hematologic neoplasias and tumor-like conditions, e.g., Hodgkin's lymphoma; non-Hodgkin's lymphomas (Burkitt's lymphoma, small lymphocytic lymphoma / chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-cell lymphoma, marginal zone lymphoma, hairy cell leukemia) Neoplasms of lymphoid precursor cells, including B-cell acute lymphoblastic leukemia / lymphoma and T-cell acute lymphoblastic leukemia / lymphoma; thymoma; peripheral T-cell leukemia; adult T-cell leukemia / T-cell lymphoma and large granular lymphocytic leukemia; neoplasms of mature T and NK cells, including Langerhans cell granulomatosis; myeloid neoplasms, such as mature AML, undifferentiated AML, acute myeloid leukemia, including acute promyelocytic leukemia, acute myelomonocytic leukemia, and acute monocytic leukemia; The compounds may be used to treat conditions including, but not limited to, chronic myeloproliferative disorders, including dysplasia, myelodysplastic syndromes, and chronic myelogenous leukemia, tumors of the central nervous system, such as brain tumors (glioma, neuroblastoma, astrocytoma, medulloblastoma, ependymoma, and retinoblastoma), solid tumors (nasopharyngeal carcinoma, basal cell carcinoma, pancreatic cancer, cholangiocarcinoma, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical cancer, ovarian cancer, primary liver or endometrial cancer, tumors of the vascular system (angiosarcoma and hemangiopericytoma)), or other cancers.

[0129] The GL-2045 compositions of the present invention can be used to treat autoimmune diseases. As used herein, the term "autoimmune disease" refers to a diverse group of over 80 diseases and conditions. In all of these diseases and conditions, the underlying problem is that the body's immune system attacks the body itself. Autoimmune diseases affect all major body tissues, including connective tissue, nerves, muscles, the endocrine system, skin, blood, and the respiratory and digestive systems. Autoimmune diseases include, for example, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and type 1 diabetes.

[0130] The disease or condition treatable using the compositions and methods of the present invention may be a hematoimmunologic process, including, but not limited to, sickle cell disease, idiopathic thrombocytopenic purpura, alloimmune / autoimmune thrombocytopenia, acquired immune thrombocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, parvovirus B19-associated erythroid aplasia, acquired anti-factor VIII autoimmunity, acquired Won-Willebrand disease, multiple myeloma and monoclonal gammopathy of undetermined significance, sepsis, aplastic anemia, erythroid aplasia, Diamond-Blackfan anemia, hemolytic disease of the newborn, immune-mediated neutropenia, resistance to platelet transfusions, post-transfusion purpura of the newborn, hemolytic uremic syndrome, systemic vasculitis, thrombotic thrombocytopenic purpura, or Evans syndrome.

[0131] The disease or condition may also be a neuroimmunological process, including, but not limited to, Guillain-Barré syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, paraproteinemic IgM demyelinating polyneuropathy, Lambert-Eaton myasthenic syndrome, myasthenia gravis, multifocal motor neuropathy, lower motor neuron syndrome associated with anti-GM1, demyelination, multiple sclerosis and optic neuritis, stiff man syndrome, paraneoplastic cerebellar degeneration with anti-Yo antibodies, paraneoplastic encephalomyelopathy, sensory neuropathy with anti-Hu antibodies, epilepsy, encephalitis, myelitis, myelopathy specifically associated with human T-cell lymphotropic virus 1, autoimmune diabetic neuropathy, Alzheimer's disease, Parkinson's disease, Huntington's disease, or acute idiopathic dysautonomia.

[0132] The disease or condition can also be inflammation or autoimmunity associated with hearing loss or blindness.For example, the disease or condition can be autoimmune-related hearing loss, such as noise-induced hearing loss or age-related hearing loss, or can be associated with the implantation of a device, such as an auditory device (e.g., cochlear implant).In some embodiments, the compositions provided herein can be administered to a subject before, simultaneously with, or after the implantation of a device.

[0133] The disease or condition may also be a rheumatic disease process, including, but not limited to, Kawasaki disease, rheumatoid arthritis, Felty's syndrome, ANCA-positive vasculitis, spontaneous polymyositis, dermatomyositis, antiphospholipid syndrome, recurrent spontaneous abortion, systemic lupus erythematosus, juvenile idiopathic arthritis, Raynaud's disease, CREST syndrome, or uveitis.

[0134] The disease or condition may also be an immunocutaneous disease process including, but not limited to, toxic epidermal necrolysis, gangrene, granulomatosis, autoimmune blistering skin diseases including pemphigus vulgaris, bullous pemphigoid, pemphigus foliaceus, vitiligo, streptococcal toxic shock syndrome, systemic sclerosis including scleroderma, extensive and extreme cutaneous systemic sclerosis, or atopic dermatitis (especially steroid-dependent).

[0135] The disease or condition may also be a musculoskeletal immunological disease process, including, but not limited to, inclusion body myositis, necrotic fasciitis, inflammatory myopathy, myositis, anti-decorin (BJ antigen) myopathy, paraneoplastic necrotizing myopathy, X-linked vacuolated myopathy, penicillamine-induced polymyositis, atherosclerosis, coronary artery disease, or cardiomyopathy.

[0136] The disease or condition may also be a gastrointestinal immunological disease process, including, but not limited to, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac disease, dermatitis herpetiformis, idiopathic cirrhosis, reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis, or sclerosing cholangitis.

[0137] The disease or condition can also be graft-versus-host disease, antibody-mediated graft rejection, post-bone marrow transplant rejection, post-infectious disease inflammation, lymphoma, leukemia, neoplasia, asthma, type 1 diabetes with anti-beta cell antibodies, Sjogren's syndrome, mixed connective tissue disease, Addison's disease, Vogt-Koyanagi-Harada syndrome, membranoproliferative glomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener's granulomatosis, micropolygyria, Churg-Strauss syndrome, polyarteritis nodosa, or multisystem organ failure.

[0138] As used herein, "allergy" includes all immune responses mediated by IgE, as well as those that mimic IgE-mediated responses. Allergies are induced by allergies that include proteins, peptides, carbohydrates, and combinations thereof that trigger IgE or IgE-like immune responses. Exemplary allergies include nut allergies, pollen allergies, and insect sting allergies. Exemplary allergies include urushiol in poison ivy and oak; house dust antigens; birch pollen components Bet v 1 and Bet v 2; the 15 kD antigen in celery; the apple antigen Mal d 1; Pru p 3 in peach; timothy grass pollen allergy Phl p 1; Lol p 3, Lol p 1, or Lol p V in rye grass; Cyn d 1 in beegrass; house dust mite Der p 1, Der p 2, or Der f 1 for house dust mite allergy; α-gliadin and γ-gliadin epitopes in gluten; bee venom phospholipase A2; and Ara h 1, Ara h 2, and Ara h 3 epitopes in peanuts.

[0139] In another embodiment, the GL-2045 compositions described herein could be utilized in a priming system in which blood is drawn from a patient and briefly contacted with optimally manufactured stradomers for a period of about one and a half to about three hours before being introduced back into the patient. In this form of cell therapy, the patient's own effector cells are exposed to optimally manufactured stradomers anchored on a matrix ex vivo in order to modulate the effector cells through exposure of the effector cells to the optimally manufactured stradomers. The blood containing the regulated effector cells is then infused back into the patient. Such a priming system could have numerous clinical and therapeutic applications.

[0140] The GL-2045 compositions described herein can also be readily applied to modify immune system responses in a variety of contexts to affect specific changes in immune response profiles. Modification or modulation of a subject's immune response refers to an increase, decrease, or a change in the proportion or components of the immune response. For example, cytokine production or secretion levels can be increased or decreased as desired by targeting complement with an appropriate combination of FcRs with stradomers designed to bind and interact with these receptors. Antibody production can also be increased or decreased; the proportion of two or more cytokines or immune cell receptors can be altered; or additional types of cytokines or antibodies can be produced.

[0141] In a preferred embodiment, a subject with an autoimmune or inflammatory disease has their immune response altered by administering to the subject a therapeutically effective amount of a GL-2045 composition described herein, wherein the therapeutically effective amount of the GL-2045 composition alters the immune response in the subject. Ideally, this invention treats the disease or condition in the subject. The altered immune response can be an increased or decreased response and can involve altered cytokine levels, including levels of any of IL-1RA and other IL-1 family members, IL-6, IL-10, IL-8, IL-23, IL-7, IL-4, IL-12, IL-13, IL-17, IL-1 receptor, TNF-α, other TNF family members and TNF receptors, IFN-α, other interferon family members and interferon receptors, or chemokine levels, including levels of any of the CCL, CXC, XC, and FAM19 chemokine family members. In a preferred embodiment, IL-6 or IL-8 is reduced in response to treatment. In particularly preferred embodiments, IL-6 and IL-8 are reduced in response to treatment, and / or IL-10 or IL-1RA are increased in response to treatment. However, the present invention is not limited by any particular mechanism of action of the described biomimetics. The altered immune response can be an altered level of autoantibodies in the subject. The altered immune response can be an altered level of autoaggressive T cells in the subject.

[0142] For example, reducing TNF-alpha production in autoimmune diseases can have a therapeutic effect. A practical application of this is anti-TNF-alpha antibody therapy (e.g., REMICADE®), which has been clinically proven to treat plaque psoriasis, rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. These autoimmune diseases have different etiologies, but share an important immunological component of the disease process related to inflammation and immune cell activity. Stradomers designed to reduce TNF-alpha production would be similarly effective in these and many other autoimmune diseases. The altered immune response profile can also be direct or indirect modulation to affect antibody production, e.g., autoantibodies targeting the subject's own tissues, or altered levels of autologous aggressive T cells in the subject. For example, multiple sclerosis is an autoimmune disorder involving autoreactive T cells that can be treated with interferon beta therapy. See, e.g., Zafranskaya M, et al., Immunology 2007 May;121(1):29-39 - Epub 2006 Dec 18. Optimally engineered stradomers designed to reduce autoreactive T cell levels may be similarly effective in multiple sclerosis and other autoimmune diseases involving autoreactive T cells.

[0143] The GL-2045 compositions described herein can be used to modulate the expression of costimulatory molecules from immune cells, including dendritic cells, macrophages, osteoclasts, monocytes, or NK cells, or to suppress the differentiation, maturation, or cytokine secretion, including interleukin-12 (IL-12), or to increase cytokine secretion, including interleukin-10 (IL-10), interleukin-6 (IL-6), or IL1-RA, of these same immune cells. One skilled in the art can also verify the efficacy of an optimized immunologically active biomimetic by exposing immune cells to the optimized immunologically active biomimetic and measuring modulation of immune cell function, where the immune cells are dendritic cells, macrophages, osteoclasts, or monocytes. In one embodiment, the method further includes exposing immune cells to the optimized immunologically active biomimetic in vitro and determining the amount of cell surface receptors or the amount of cytokine production, where a change in the amount of cell surface receptors or cytokine production indicates modulation of immune cell function. In another embodiment, the method further comprises exposing immune cells in vivo to the optimized immunologically active biomimetic in an animal model for autoimmune disease and assessing the extent of improvement in the autoimmune disease.

[0144] The GL-2045 compositions described herein can also be used as components of devices. In some embodiments, the GL-2045 provided herein can be coated onto devices such as medical implants. For example, optimally engineered stradomers can be coated onto coronary stents or as part of nanoparticle therapy to improve penetration and prolong drug release, for intraocular use in, for example, uveitis or macular degeneration. The optimally engineered stradomers described herein can also be used as a component of diagnostics. In some embodiments, one skilled in the art can personalize treatment by determining which patients would particularly benefit from the use of stradomers. For example, one skilled in the art can expose a patient's immune cells to an immunologically active biomimetic and measure modulation of immune cell activation or maturation by flow cytometry or cytokine profiling to identify high responders.

[0145] All references cited herein are incorporated by reference in their entirety. [Example]

[0146] Various approaches were taken in the manufacturing process to optimize the combination of high protein titer, long viability with associated low cell debris, and production of higher order multimers of GL-2045. Specifically, the following aspects of the upstream manufacturing process were varied to determine optimal conditions for GL-2045 production with increased multimerization characteristics: basal medium, feed type, feed timing, temperature shift, aeration, and shake flask conditions. In each example, cell density, viability, protein titer, and multimerization were analyzed to identify optimal conditions. Additionally, aspects of the downstream manufacturing process were varied, including buffers, wash protocols, and column selection, to determine optimal conditions for purification and filtration of GL-2045 that maintained the optimal multimerization profile of GL-2045. The following examples are offered by way of illustration only and not by way of limitation.

[0147] Example 1 - Fractionation and Biolayer Interferometry Analysis of GL-2045 The GL-2045 solution was fractionated using a GE Hi-Load 26 / 60 Superdex 200 pg column (GE, #17-1071-01) in 0.05 M Tris-HCl + 0.15 M NaCl buffer (pH 7.5). 3.2 mL of the GL-2045 solution was loaded at a flow rate of 2.6 mL / min. Six fractions (1–6) were collected in 2.0 mL volumes, and protein concentrations were determined by UV measurement at 280 nm (Figure 1A). Multimerization was assessed for each of the GL-2045 fractions. Briefly, samples from fractions 1–6 were loaded onto 4–12% non-reducing Nu-Page BT gels (Invitrogen, #NP0322BOX). Samples were run at 150 volts for approximately 3 hours. The results are presented in Figure 1B and demonstrate a clear difference in the presence of higher order multimers of GL-2045 in fractions 1-6. Fractions 1-3 are composed of lower order multimers (e.g., bands 1-4). Fraction 1 is composed almost exclusively of homodimers with an apparent MW of 55 KD (band 1, MW estimated from non-reducing SDS-PAGE). Fraction 2 is composed of approximately 97% homodimers with a MW of 110 KD (band 2). Fraction 3 is composed primarily of bands 3 and 4 with MWs of 165 KD and 220 KD, respectively, with lesser amounts of bands 2, 5 (MW = 275 KD), 6 (MW = 330 KD), and 7 (MW = 385 KD). Fraction 4 is composed primarily of bands 4, 5, and 6, along with lesser amounts of bands 3, 7, 8 (MW=440 KD) and higher order bands. Fractions 5 and 6, however, are composed primarily of higher order multimers (band 5+).

[0148] A fraction of GL-2045 was analyzed for binding to the FcγRIIIA receptor using biolayer interferometry kinetic binding analysis. Biolayer interferometry detects binding between a ligand immobilized on a biosensor chip surface and an analyte in solution. Binding generates an increase in optical thickness at the biosensor chip, resulting in a wavelength shift (detected as a response unit, "RU"). The maximum binding level (RU max) is the maximum possible amount of sample binding at equilibrium that saturates the amount of ligand on the sensor surface.

[0149] His-tagged receptor protein (5 μg / mL) was bound to an anti-His sensor chip (anti-Penta-His HIS1K, ForteBio Cat. #18-5121) in 1× kinetic analysis buffer from ForteBio (Cat. #18-1092) for 300 seconds. The loaded sensor was transferred to 1× kinetic buffer without labeled receptor or ligand to obtain a baseline measurement for 60 seconds. After obtaining the baseline, the receptor / protein on-ratio was measured by transferring the sensor chip to 1× kinetic buffer containing selected purified stradomers at concentrations of 50 μg / mL, 25 μg / mL, and 12.5 μg / mL for 60 seconds. The off-ratio was measured by transferring the sensor chip to 1× kinetic buffer for 300 seconds, and RU values, on-ratio values, dissociation ratios, and K values ​​were recorded. d Values ​​were calculated using ForteBio software.

[0150] Binding curve results are shown in Figure 2, and kinetic binding data calculated by ForteBio Octet software are provided in Table 2. These binding curves demonstrate higher binding avidity, with increasingly lower off ratios, for fractions containing higher molecular weight GL-2045 (e.g., fractions 3, 4, 5, and 6) than that observed for lower molecular weight fractions (e.g., fractions 1 and 2), indicating that the higher molecular weight fractions of GL-2045 bind more avidly than the lower molecular weight fractions. [Table 2]

[0151] Example 2 - Complement-dependent cell killing (CDC) assay using GL-2045 fractions The ability of GL-2045 fractions to inhibit complement activation was evaluated. GL-2045 was fractionated into six fractions by size-exclusion chromatography (Figure 3A), and each fraction was analyzed for multimerization on a non-reducing gel (Figure 3B). To determine the effect of each fraction on complement activation, CD20-expressing Wil2 cells were incubated with an anti-CD20 monoclonal antibody for 20 minutes, after which the cells were centrifuged and resuspended in fresh medium. The cells were then cultured in 96-well plates containing each of fractions 1-6 described herein, as well as unfractionated GL-2045 as a comparison at one of six concentrations: 100 μg / mL, 50 μg / mL, 20 μg / mL, 10 μg / mL, 5 μg / mL, or 1 μg / mL. To initiate complement-dependent cell lysis, serum was added to the cell suspension, and the plates were incubated at 37°C for 3 hours. Cell death was quantified using the Promega Cytotox Glo assay. Cytotox assay reagent was added to each well of the plate, and the plate was incubated in the dark at room temperature for 15 minutes. Luminescence after 15 minutes was read on a Promega GloMax luminometer, and cell death was calculated from this reading. The results are shown in Figures 4A-4D and demonstrate that fractions 5 and 6 (containing higher molecular weight multimers in bands 5-13) exhibit more pronounced inhibition of CDC than the lower molecular weight multimers present in fractions 1-4. It is also noted that only fractions containing four or more bands demonstrate effective inhibition of CDC, consistent with multivalent Fc binding of higher order multimers to hexameric C1q.

[0152] Example 3 - Binding of GL-2045 fractions to FcγRIIIa The binding of GL-2045 fractions to FcγRIIIa was determined. GL-2045 supernatant was purified by affinity chromatography with Protein A HiTrap MabSelect Sure (GE#11-0034-95) using a binding buffer of 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2, and eluted with 0.1 M glycine, pH 2.7 (FIG. 5A). Affinity chromatography-purified GL-2045 was stored in 1× PBS (Quality Biological, Inc. #119-069-101), pH 7.2. The purified GL-2045 pool was further dialyzed against 50 mM sodium acetate, pH 5.0, and purified by cation exchange chromatography on a POROS CIEX column (1.2 cmD x 10 cmL GOPURE column, Poros XS, Life Technologies, #4448885) using a binding buffer of 50 mM sodium acetate, pH 5.0, and an elution gradient (0-100% elution buffer) of 1 M NaCl, 50 mM sodium acetate, pH 5.0. Polish This Polish The steps were performed without removing the highest order multimers and / or disorganized aggregates from the final fraction. Polish The extracted GL-2045 was concentrated to a volume of ≦5 mL, buffer exchanged into gel filtration running buffer, and loaded onto a gel filtration column (Hiload 26 / 60 Superdex 200 pg (GE #17-1071-01)) using 0.05 M Tris-HCl + 0.15 M NaCl pH 7.5 as the running buffer (Tris HCl, pH 7.5 Teknova #T1075). Fractions were then analyzed for multimerization by gel analysis (FIGS. 5B and 5C).

[0153] The binding of fractionated GL-2045 to FcγRIIIa was determined using an FcγRIIIa ELISA binding assay. Briefly, 96-well plates were coated with recombinant FcγRIIIa and reacted with GL-2045. After washing, the amount of FcγRIIIa-bound material was determined using an Fc detection mAb in an ELISA-based assay (Figures 6A and 6B). The EC for each fraction was 0.01. 50 The values ​​are shown in Table 3. These results indicate that the higher order multimers (fractions 1C4, 1C5, 1C6, 1C7, 1C8, 1C9, 1C10, and 1C11) had lower EC for FcγRIIIa than the lower order multimers (fractions 1D9, 1E4, and 1F7). 50 It is noted that the GL-2045 fractions demonstrate more avid binding and, surprisingly, demonstrate more avid binding than the highest molecular weight multimers (fractions 1C3, 1C2, 1C1, 1B12, 1B11). The very highest molecular weight fractions are predicated on containing certain lower potency high molecular weight aggregated fractions along with the highly functional highest order multimers (e.g., fractions 1B11, 1B12, 1C1, 1C2, and 1C3). These results, quite surprisingly, indicate that not all high molecular weight fractions of GL-2045 demonstrate increased binding to FcγRs, likely due to the effect of disordered assembly of homodimers as opposed to the formation of highly ordered high molecular weight multimers. These data support the generation and retention of higher molecular weight, even higher order multimers and the less effective binding to the lower affinity receptors of the binding target (EC values ​​for 1B11 and 1B12 in Table 3). 50 (See values) demonstrate the need for optimized downstream manufacturing methods (including optimized conditions for Protein A purification, ion exchange chromatography, and hydrophobic interaction chromatography) in combination with optimal upstream manufacturing methods to result in the removal of unordered high molecular weight aggregates (e.g., 1B11 and 1B12). [Table 3]

[0154] Example 4 - C5a-induced chemotaxis analysis of GL-2045 fractions GL-2045 cell cultures were grown in PowerCHO2 medium (Lonza, #U21-070) with L-glutamine (Lonza, #17-605E) and HT supplement (Life Technologies, #11067-030). GL-2045 supernatants were purified by affinity chromatography using Protein A HiTrap MabSelect Sure (GE, #11-0034-95) and then fractionated on an AIEX HiScreen Q FF (GE, #28-9505-10) using different pH conditions to separate low-molecular-weight bands from high-molecular-weight bands. The results are shown in Figure 7 (GL-GLM-01 = recombinant, unfractionated Fc (G001), GL-GLM-02 = unfractionated GL-2045, GL-GLM-05 = fractionated GL-2045 at pH 6.0, GL-GLM-06 = fractionated GL-2045 at pH 6.5, GL-GLM-07 = fractionated GL-2045 at pH 7.0, GL-GLM-08 = fractionated GL-2045 at pH 7.5). Finally, the different fractions were concentrated and dialyzed against HBSS (Lonza, #10-527F).

[0155] The supernatant of GL-2045 was purified by affinity chromatography with protein A (pA) HiTrap MabSelect Sure (GE, #11-0034-95) using a binding buffer of 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2. After a first wash with the binding buffer and a second wash with a buffer containing 1 M NaCl, 5 mM EDTA, 2 M urea, 50 mM phosphate, pH 7.0, the protein bound to pA was eluted with 0.1 M glycine, pH 2.7.

[0156] Affinity chromatography-purified GL-2045 was stored in 1X PBS (Quality Biological, Inc. #119-069-101) at pH 7.0. Four batches of purified GL-2045 were further diluted (6X) with 50 mM Tris-HCl at pH 6.0, 6.5, 7.0, or 7.5 and purified by anion exchange chromatography on a HiScreen Q FF column using a binding buffer of 50 mM Tris-HCl at pH 6.0, 6.5, 7.0, or 7.5, and eluted by gradient elution (0-100% elution buffer) using 50 mM Tris-HCl + 1 M NaCl at pH 6.0, 6.5, 7.0, or 7.5.

[0157] These purified fractions were used to determine the effect of GL-2045 fractions on neutrophil chemotaxis. Briefly, complement C5a was added as a chemoattractant to the lower well of the Boyden chamber at a concentration of 1 nM. Neutrophils (purified from whole blood from PBMCs, final concentration 2.25 x 10) were added to the Boyden chamber prior to addition. 6 Cells (1000 cells / mL) were pre-incubated for 30 min with the indicated GL-2045 fractions (final concentrations of 0.02-10 µg / mL) or recombinant Fc control (GLM-001, G001). The cell suspension was then added to the upper well of the Boyden chamber and incubated for 25 min. After the incubation period, the migrated population was assessed by counting the number of cells in the lower chamber for each condition, and the percent chemotaxis for each condition was determined (Figure 8). No chemotaxis was observed for GL-GLM-001 (recombinant Fc control, G001). Higher-order multimers (including bands 5–13, fractions GL-GLM-002, GL-GLM-005, GL-GLM-006, and GL-GLM-007) demonstrated more phagocytic inhibition of C5a-induced chemotaxis than lower-order multimers (including bands 1–4, fraction GL-GLM-008).

[0158] The data provided in Examples 1-4 demonstrate the enhanced potency of higher order multimers for GL-2045. Based on the above data, upstream manufacturing protocols were tested to determine optimal conditions for specific production of higher order multimers of GL-2045 (e.g., band 5+ in Figures 1, 3, 5, and 7) to achieve maximum biological potency.

[0159] Example 5 - Screening of basal media for GL-2045 production The purpose of this experiment was to test the effect of a panel of basal media on GL-2045 protein titer, cell viability, cell density, and GL-2045 multimerization.

[0160] GL-2045 was grown in ProCHO5 medium (Lonza #12-766Q) containing L-glutamine (Lonza #17-605E) and hypoxanthine sodium and thymidine (HT, Gibco #11067-030) in a shaker incubator at 37°C and 5% CO. After passage, cells were washed and plated at 0.5 x 10 in the selected medium (shown in Table 4) in a 50 mL tube spin. 6 The cells were inoculated in duplicate at 0.1% cells / mL. Each tube contained 10 mL of culture and was placed in a Kuhner brand magnetic levitation shaker incubator at 37°C, 5% CO2, 80% humidity, and 180 rpm. On days 4, 8, and 10, 1 milliliter samples were removed from each culture to measure cell density, cell viability, and glucose levels. Samples were centrifuged, and the supernatants were stored at +4°C. Selected media not listed were supplemented with 4 mM L-glutamine and 1X hypoxanthine sodium and thymidine as ingredients. Growth conditions for selected media are shown in Table 4. [Table 4]

[0161] GL-2045 cell cultures grown in selected media were evaluated for cell density, cell viability, protein titer, and percentage of higher-order multimers. Cell density and cell viability were assessed by mixing the cells with trypan blue. A manual cell counter was used to count live and dead cells. Data for cell density (Figure 9, Table 5) and cell viability (Figure 10, Table 6) are shown for days 4 and 8 of culture. Of the 19 media tested, maximum cell density was observed on day 4 with ActiCHO P, CHOMACS CD, and CD FortiCHO. At day 8, the cell density trends were negative compared to day 4 for all media except ActiCHO P, BalanCD CHO, ExpiCHO, Cell Vento CHO 210, and Cell Vento CHO 210. Of the 19 media tested, ActiCHO P showed the highest cell viability at day 8, followed by ExpiCHO, Cell Vento CHO 110, and HYQ SFX-CHO LM. Of the 19 media, ActiCHO P was the only medium with a positive trend in cell viability from days 4 to 8. Thus, ActiCHO P is the only medium that produces high cell density at day 4 and does not have a negative trend for cell density at day 8. [Table 5] [Table 6]

[0162] The culture was pelleted on day 10, 0.2 μm filtered, and kept at 4°C until purified using a protein A affinity column. For purification, the supernatant culture was purified by affinity chromatography with a 1 mL protein A column HiTrap MabSelect SuRe (GE, #11-0034-93) containing a binding buffer of 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2. The column-bound protein was washed with binding buffer, followed by a second wash with 1 M NaCl, 5 mM EDTA, 2 M urea, 50 mM phosphate, pH 7.0. Column-bound GL-2045 was eluted with 0.1 M glycine, pH 2.7, and desalted in 1X PBS, pH 7.0, through a HiPrep 26 / 10 desalting column (GE, #17-5087-01). All purified samples were stored at 4°C. Protein titer measurements were performed by biolayer interferometry (Octet) (Figure 11 and Table 7). Of the 19 basal media tested, the highest protein titer at day 10 was observed with Cell Vento CHO 110, followed by BalanCD CHO Growth A medium, ActiCHO P, and ExpiCHO. A significant decrease in titer occurred with the other media. [Table 7]

[0163] To determine the proportion of higher-order multimers, each purified culture was run (in unreduced form) on an SDS-PAGE gel (NuPage 3-8% Tris-Acetate Gel, Life Technologies, #EA03752BOX). 2 μg of protein was diluted to a final volume of 10 μL in 3 μL of sample buffer (NuPage LDS (4X), Life Technologies, #NP0007), 20 μM iodoacetamide (Bio Rad #163-2109), and deionized (DI) water. The sample was heated at 80°C for 10 minutes, loaded onto the gel, and run at 150 V for 1 hour and 25 minutes using running buffer (Tris-Acetate SDS (20X) (Life Technologies, #LA0041)). The gel was washed in DI water, stained with SimplyBlue Safe (Life Technologies, #LC6060), and destained with DI water. After complete destaining, images were cropped using a Syngene G:BOX system, and the band patterns were analyzed by densitometry using the GeneTool software of Syngene. The intensity of each individual band in each lane was measured (Figures 12A and 12B).

[0164] Unexpectedly, we observed the highest percentage of higher order multimers (>4 bands) among the 19 media tested with ActiCHO P, followed by EX-CELL CD CHO and CH-S SFM2 (Table 8). These data indicate that the increase in the percentage of higher order multimers is an independent variable that should be controlled and does not simply correlate with an increase in total protein titer. ActiCHO P produced the third highest protein titer and the highest level of multimerization (45.9% of the protein present in band 5+), while Cell Vento CHO 110 produced the highest protein titer but a substantially lower level of multimerization (32.6% of the protein present in band 5+). [Table 8]

[0165] Example 6 - Screening of GL-2045 media by feeding Recommended Feeding Schedule Based on the results of the experiment in Example 5, the four basal media associated with the highest GL-2045 protein titers and the three basal media associated with the lowest GL-2045 protein titers were subjected to replicate experiments in which commercially available feeds were provided during culture. Cell Vento 110 and ExpiCHO, which produced high titers, were not selected for feeding experiments because the manufacturer's recommended feeds were not identified. Cell Vento CHO 110 is a complete medium to be used for cell adaptation without feeding, while Cell Vento 210 is used for culture in combination with feeding. The media and feed combinations used in this experiment are detailed in Table 9. [Table 9]

[0166] GL-2045 clone 58 was cultured in "ProCHO5" medium (Lonza #12-766Q) with L-glutamine (Lonza #17-605E) and hypoxanthine sodium and thymidine (HT, Gibco #11067-030) in a shaker incubator at 37°C and 5% CO2. After passage, cells were washed and diluted to 0.5 x 10 6 cells / ml, then inoculated at a density of 1 × 10 6 ~3×10 6 Cells were subcultured when they reached 0.5x10 cells / mL and ≥80% viability. GL-2045 clone 58 was adapted directly to the selected medium detailed in Table 10. Adaptation was considered complete when a stable doubling time (20-30 hours) and ≥90% viable cell density (VCD) was obtained for at least 2-3 passages. Cells were cultured at 0.5x10 cells / mL in the selected medium (d0). 6Cells were seeded at 0.1 cells / mL and cultured in a standard shaking platform in an incubator set at 150 rpm at 37°C, 5% CO2, and high humidity. Feeding began on day 3 (d3) for all cultures except PowerCHO3 CD. Feeding for PowerCHO3 CD medium began on day 1. The total culture volume was 120 mL. Cultures were harvested when viability dropped to 50%. GL-2045 stable cell lines were grown in each of the seven media with the manufacturer's recommended feeds according to the recommended protocol. The feeding strategy and schedule for each of the media tested are outlined in Figures 13A and 13B.

[0167] Measurements of cell density, cell viability, and GL-2045 protein titer were performed throughout the study. For protein titer, samples were centrifuged to pellet cells, and protein in the cell supernatant was measured by biolayer interferometry (Octet) of the cell supernatant as described in Example 6. GL-2045 multimerization was assessed at the end of each arm of the study after Protein A purification as described in Example 5. Cultures were continued on day 14 or until viability fell below 50%.

[0168] Surprisingly, GL-2045 grown in ActiCHO P with the manufacturer's recommended feeding achieved a much higher peak cell density than GL-2045 grown in any of the other media with the manufacturer's recommended feeding. This superior cell density was surprisingly more than three times higher than all other media / feed combinations tested, except for Cell Vento 210 (Figure 14).

[0169] Furthermore, GL-2045 grown in ActiCHO P, CD FortiCHO, or ADCF MAB with the manufacturer's recommended feeding achieved much better (2-3 fold) cell viability at day 10 than GL-2045 grown with the manufacturer's recommended feeding in the other media tested, demonstrating that these three media produce superior cell viability compared to several other media / feed combinations tested (Figure 15).

[0170] In addition, the GL-2045 CHO stable cell line grown in ActiCHO P medium with the manufacturer's recommended ActiCHO diets A and B developed substantially higher titers than any other medium and manufacturer-recommended feed tested ( FIG. 16 ). Titers generated from ActiCHO P cultures were at least four-fold higher than titers generated from any of the other media tested, reaching 2 g / L on day 10 in shake flasks, compared to less than 500 mg / L for Cell Vento CHO-210 on day 12 and even less for the other media.

[0171] GL-2045 multimerization, as measured by the percentage multimers in bands 5 or higher (5+), under different culture conditions was determined as described in Example 5 (Figure 17, Table 10). The highest percentage of GL-2045 multimerization using the manufacturer's recommended basal medium and feed was with CD FortiCHO, followed by ActiCHO and PowerCHO3. ADCF-Mab and BalanCD demonstrated significantly worse GL-2045 multimerization compared to other culture conditions. [Table 10]

[0172] In summary, ActiCHO P medium is significantly better than the other media tested with respect to cell density, viability, and protein titer. Densitometry analysis shows that CD FortiCHO medium + CD Efficient Feed C has the highest percentage of maximally active multimers at 35.7% (Table 10, band 5+). However, CD FortiCHO medium + CD Efficient Feed C has the highest percentage of polymeric material that does not migrate in the gel at 4.7% (as seen at the top of the gel in Figure 17), suggesting that this medium also generates a larger fraction of aggregated, unordered multimers with less highly ordered and more functional multimers than ActiCHO P. ActiCHO P + Feeds A and B have a second highest percentage of an even higher-order multimer band above band 4 at 30.1%, with negligible amounts of nonspecifically aggregated, high-molecular-weight material that does not migrate in the gel. Thus, ActiCHO-P likely has the highest percentage of fully functional higher order multimer bands. These data further demonstrate that upstream manufacturing conditions not only affect cell viability, density, and total protein titer, but also the generation of clinically effective higher order multimers of GL-2045. As demonstrated in Example 3, the highest molecular weight fractions exhibited reduced binding to FcRγIIIa, suggesting that the highest molecular weight fractions (e.g., disordered GL-2045 aggregates) are less biologically active than highly ordered GL-2045 multimers. As shown herein, quite surprisingly, of all the basal media tested, only ActiCHO-P demonstrated high total GL-2045 protein titer, high multimerization, and minimal amounts of disordered, high molecular weight GL-2045 aggregates.

[0173] Modified feeding protocol After determining that ActiCHO P medium + feeding resulted in optimal protein titers and production of higher multimers, an altered feeding schedule was tested to determine whether similar or optimized results could be obtained by feeding every other day. As demonstrated in Figure 18, feeding every other day (blue) did not significantly affect cell density (Figure 18A), cell viability (Figure 18B), culture pH (Figure 18C), or protein titer (Figure 18D) compared to daily feeding. These results surprisingly indicate that feeding every other day can achieve similar results and may be preferable for maintaining sterility and minimizing production costs. However, similar experiments with feeding every third day suggested that viability and protein production may begin to decline with feeding less frequently than every other day (Figure 19).

[0174] Additional experiments were performed to determine whether ActiCHO P feeding could be used with other non-ActiCHO basal media to achieve similar results. Briefly, GL-2045 clone 58 was cultured in Power CHO-2 CD (Lonza, #12-771Q) + 4 mM L-glutamine (Lonza, #17-605E) + 1X HT supplement (Gibco, #11067-030). After passage, cells were washed and plated at 0.3 x 10 cells / ml in ActiCHO P complete medium, ActiCHO P + 6 mM L-glutamine (Lonza, #17-605E) or Power CHO-2 CD complete medium. 6ActiCHO P PowerCHO cultures were inoculated at 1000 cells / ml and cultured without temperature shift. ActiCHO P PowerCHO cultures were fed daily with +1 mL of ActiCHO feed A (PAA, #U15-072) + 0.1 mL of feed B (PAA, #U05-034). On day 9, cell viability and protein titer were determined as described in Example 5 throughout the culture (Figure 20). The results show that both PowerCHO and ActiCHO P generate similar cell viability and GL-2045 protein yields when used with ActiCHO feeds A and B according to the manufacturer's recommendations. Therefore, ActiCHO feeds A and B may be able to be used with other selective high-performing basal media if the multimer composition remains unchanged for ActiCHO P+A+B.

[0175] Example 7 - Optimal Timing and Extension of Temperature Shift for GL-2045 Production The purpose of this experiment was to evaluate the optimal timing and duration of the temperature shift. Numerous investigators have examined the effects of temperature shift on the cell cycle, apoptosis, and metabolism of recombinant Chinese hamster ovary (CHO) cell lines. However, consideration has generally been given to the effect of temperature shift on viable cell density, with little, if any, attention paid to the minimum cell density required for optimal temperature shift results. Furthermore, the minimum cell density required for successful temperature shift in multimerized stradomers has not been examined.

[0176] The present inventors surprisingly found that optimal GL-2045 expression and maximum titer require a minimum viable cell density of 10 million cells / mL at the time of temperature shift. Furthermore, this requirement is more critical than the day of culture on which the temperature shift occurs. Surprisingly, the timing of the temperature shift is most successful when the viable cell density is in the logarithmic growth phase, typically between 10 and 15 million cells / mL. This generally corresponds to day 4 or 5 of bioreactor culture, depending on the initial seeding density.

[0177] Further deviation from what is described in the art, for optimal upstream conditions for producing high-titer GL-2045, a temperature shift of 37 degrees Celsius (37°C) + / - 1.0°C to 32.5°C + / - 1.0°C is preferred over a temperature shift to 31°C + / - 0.5°C. Two separate pools of GL-2045 supernatant were generated from stable CHO clones using identical conditions, except for the nature of the temperature shift. CHO cells were cultured in a 10 L XDR-10 Single-Use Bioreactor System bioreactor (Xcellerex, GE) with a pH shift of 7.1 to 7.0 on day 5. Seven liters of ActiCHO-P CD (cat#U21-051) were used for production, along with 280 ml of PAA Feed A (cat#U15-053) daily and 28 ml of PAA Feed B (cat#U15-054) daily. PAA Diets A and B are equivalent to ActiCHO Diets A and B. Temperature shifts to 32.5°C (A) and 31.0°C (B), respectively, occurred on day 5. Results are shown in Table 11. [Table 11]

[0178] Example 8 - Protein A column purification of GL-2045 requires more frequent CIP procedures GL-2045 was purified by affinity chromatography (AC) using a Protein A HiTrap MabSelect SuRe column (GE#11-0034-95) with a binding buffer of 20 mM sodium phosphate, pH 7.2, 0.15 M NaCl, and eluted with 0.1 M glycine, pH 2.7. AC-purified GL-2045 was stored in 1X PBS, pH 7.0 (Quality Biological, Inc. #119-069-101). At the end of each experiment, AC GL-2045 purification was processed without a column clean-in-place (CIP) procedure. The CIP procedure typically involves running dilute sodium hydroxide (0.1–0.5 M NaOH) through the column between purification cycles to sanitize the Protein A resin while hydrolyzing precipitates, thereby regenerating the binding capacity of the Protein A column (Boulet-Audet et al., Scientific Reports, Vol. 6, 2016). GL-2045 was purified on four separate Protein A affinity columns (columns 1–4, run 11.27.12). The same Protein A affinity column was then used for a second affinity purification run (run 11.28.12) after eluting the column with 100% elution buffer. Two purifications of GL2045 using the same column showed reduced amounts of lower molecular weight species, both homodimers and homodimeric dimers, after the second purification run (Figure 22). As shown in Table 12, densitometry analysis of SDS-PAGE demonstrated a significant loss of homodimer and dimer bands, indicating that the lower molecular weight bands are exceeded in binding to the Protein A affinity column by the remaining highly Protein A-greedy GL-2045 protein, which is not completely removed from the Protein A column by elution with the elution buffer. [Table 12]

[0179] The loss of lower molecular weight bands indicates that high molecular weight multimeric GL-2045, containing multiple Protein A binding sites, outweighs lower molecular weight species, resulting in the loss of lower molecular weight species and effectively altering the composition of the drug, indicating avidity-based binding to the affinity column. These data suggest that more frequent CIP procedures, and thus more frequent regeneration of the Protein A column, are necessary for optimal purification of GL-2045 when using a Protein A column for multiple purification runs. These results were unexpected because, as described above, regeneration typically requires the use of NaOH, which would degrade the Protein A columns most commonly used in the art. Therefore, more frequent use of such buffers would result in more rapid degradation of the Protein A column. Thus, a Protein A column that can withstand frequent CIP procedures with a high-strength NaOH buffer, such as 0.5 M NaOH, must be used to regenerate a Protein A column used repeatedly in the purification of GL-2045 to maintain the optimal profile of the intact GL-2045 drug.

[0180] Example 9 - CIP procedure to regenerate Protein A for repeated purification cycles of GL-2045 requires 0.5M NaOH The loss of low molecular weight species in the absence of a CIP step, as shown in Example 8, also indicates that there are high molecular weight species that remain bound to the Protein A affinity column after the first column run, thereby occupying binding sites and preventing binding of lower molecular weight species in subsequent purification runs. These data indicated that an additional CIP step should be used to maintain the multimeric profile of Protein A-purified GL-2045.

[0181] We found that routine daily purification using a HiTrap MabSelect column (GE #28-4082-58) resulted in a significant change in the composition of purified GL-2045 after approximately 6–7 purification runs, due to a slight loss of homodimer and dimer fractions. For example, a skilled practitioner purifying monoclonal antibodies would not expect to find a change in the composition of the purified product after 6–7 Protein A purification runs. To address this issue, we performed the manufacturer-recommended CIP procedure with 0.1 M NaOH to regenerate the binding capacity of the Protein A column. These CIP procedures were performed after each purification run, which is more frequent than is commonly used in the art. While frequent CIP procedures provided some improvement, they did not resolve the problem of loss of lower molecular weight species. We therefore reasoned that the avid binding of GL-2045 to protein A, to promote retention of homodimers and dimers, required a more stringent CIP regimen than one skilled in the art would typically use for complete regeneration of the column.

[0182] However, resins commonly used in Protein A columns (e.g., MabSelect) are not NaOH-resistant and therefore rapidly deteriorate with the use of more stringent NaOH buffers, and such regeneration is associated with reduced purification capacity and changes in the composition of GL-2045 multimers. However, less commonly used Protein A media (e.g., MabSelect SuRe (General Electric #11-0034-95)) can withstand improved cleaning with 0.5 M NaOH. Thus, we used a MabSelect SuRe column with a daily CIP procedure using 0.5 M NaOH buffer. After performing the more frequent and more stringent CIP procedure, we achieved Protein A purification of GL-2045 on a daily basis without loss of homodimers or dimers and without changes in the composition of GL-2045.

[0183] Thus, the inventors have discovered that Protein A column CIP of GL-2045 requires more stringent and more frequent CIP procedures than would normally be used by those skilled in the art working with monoclonal antibodies or Fc fusion proteins in order to preserve the optimal profile of homodimers and dimers, and thus intact GL-2045 drug.

[0184] Example 10 - Purification of GL-2045 in homodimeric, non-highly ordered aggregates using a pH elution gradient on a Protein A column pH elution gradients are commonly used with Protein A columns during protein purification to optimize total protein yield, but are not typically used to alter the composition of the drug. We discovered that such pH elution gradients on Protein A columns can be used to remove disordered aggregates of GL-2045 from higher-order multimers. GL-2045 CHO supernatant was purified by affinity chromatography (AC) with a Protein A HiTrap MabSelect SuRe (GE#11-0034-95) containing a mixed buffer of 20 mM sodium phosphate, 0.15 M NaCl, pH 7.2, followed by an additional wash with binding buffer. Protein A-bound GL-2045 was eluted with an elution buffer containing 0% to 100% 0.1 M glycine, pH 2.7, thereby creating a pH gradient for elution of GL-2045 from the Protein A affinity column. The eluted fractions were collected into a 96-well plate (Greiner Bio-One #780271) and neutralized to pH 7.5 by adding Tris-HCl, pH 9.0, to each well. The equivalent protein amount of each fraction was then analyzed on a non-reducing SDS-PAGE gel (4-12% NuPage Bis-Tris, Invitrogen #NPO322BOX).

[0185] SDS-PAGE analysis of fractions obtained by elution of the Protein A column with a pH elution gradient demonstrated that very high molecular weight species were eluted last in fractions D6 and D7 (Figures 23A and 23B). Surprisingly, the very first fraction (D1) also contained high molecular weight species. Thus, these results indicate that high molecular weight fractions can be separated from the major species of GL-2045 (e.g., homodimers, dimers, and higher multimers) by pH gradient elution of the Protein A affinity column. As shown in Examples 1-3, these fractions may represent less active species, as evidenced by reduced Fc receptor binding and reduced CDC inhibition.

[0186] Also shown on the gel (Figure 23B, far right lane) is the very high molecular weight fraction obtained by renaturation (CIP with 0.5 M NaOH, neutralized with HCl). These results indicate that there are high molecular weight species present on the Protein A column after elution, again demonstrating the need for a highly stringent NaOH buffer during the CIP procedure to regenerate full column binding capacity.

[0187] Thus, although not typically used for this purpose, a pH elution gradient can be used to separate and remove high molecular weight disordered aggregates of GL-2045 from biologically active lower and higher order multimers. Such separation can be used to further optimize or maintain the multimer profile of purified GL-2045. Alternatively, a step elution gradient can be used to arrive at an optimized GL-2045 composition.

[0188] Example 11 - Ion exchange column salt and pH conditions denature GL-2045 multimer compositions An important goal for the purification of GL-2045 is strict control of the multimeric composition of the final purified product. Ion exchange columns (e.g., cation and anion exchange) are used for the purification of monoclonal antibodies and Fc fusion proteins. PolishWe tested a cation exchange medium, POROS CIEX (Invitrogen GoPure XS (10 mL) cat# 4448885), using elution buffers containing different concentrations of salt. First, GL-2045 was purified by protein A affinity chromatography, then pooled and dialyzed into 50 mM sodium acetate, pH 5.0, before loading onto the CIEX column. 50 mM sodium acetate, pH 5, was used as the equilibration and wash buffer. The elution buffer for GL-2045 was 50 mM sodium acetate with variable amounts of added buffer B (1 M NaCl, pH 5, shown as % in Figure 24). Polish The effect of elution buffer (EB) salt content on chromatographic run performance was examined. Chromatographic runs were performed on an Akta Avant. Briefly, the Avant procedure involved equilibrating a CIEX column with 50 mM sodium acetate, pH 5, at 2 mL / min for a total volume of 10 column volumes (cv). 65–100 mg of GL-2045, previously dialyzed into 50 mM sodium acetate, pH 5, was loaded onto the column. The column was washed with 5 cv of binding buffer at 2 mL / min. GL-2045 was then eluted at 2 mL / min with 9–15 cv of buffer containing various percentages of NaCl (e.g., elution buffer composed of 30–50% buffer B).

[0189] The percent recovery of GL-2045 was determined for GL-2045 eluted with elution buffers containing 30% buffer b, 40% buffer b, and 50% buffer b (30% EB, 40% EB, and 50% EB, respectively) to determine the optimal range of salt concentrations for the elution buffer ( FIG. 24 ). These data demonstrated that altering the salt content in the elution buffer can substantially denature the multimeric composition of GL-2045. [Table 13]

[0190] As shown in Example 11 and quantified in Table 13, SDS-PAGE analysis demonstrated dramatic differences in the resolution of GL-2045 molecular weight species when eluted with 30% EB, 40% EB, or 50% EB elution buffer. 91.7% of the material was eluted with elution buffer containing 30-40% Buffer B, and only high molecular weight material was recovered with elution buffer containing 50% Buffer B. These data demonstrate that changing the elution buffer can substantially alter the multimeric composition of GL-2045. It is therefore clear that the elution buffer selected for ion exchange can be used to alter the multimeric composition of GL-2045, a novel use of this technology. At the same time, we believe that changes in the composition of GL-2045 are due to the ion exchange. Polish If not desired in the process, a level of precision not normally practiced by those skilled in the art may be required for ion exchange. Polish It has been found that this is necessary in the selection of the salt concentration of the elution buffer to be used in the process.

[0191] Example 12 - Ion exchange chromatography can be used to reduce or remove homodimers or dimers of homodimers. Since 91.7% of the GL-2045 material in Example 11 was eluted with an elution buffer containing 30-40% Buffer B, and the only material recovered with an elution buffer containing 50% Buffer B was high molecular weight material, an elution buffer with a range of 30-40% Buffer B was selected for further analysis. The method used was similar to that described in Example 11.

[0192] SDS-PAGE analysis demonstrated dramatic differences in the resolution of GL-2045 homodimers when eluted with elution buffers containing 35%, 36%, or 37% or more of buffer b (e.g., 35% EB, 36% EB, and 37% EB, respectively). Note that the homodimers and homodimer dimers clearly visible at 35% EB are, in contrast, greatly reduced at 36% EB and completely eliminated at 37% EB or higher (Figure 25). Similarly, SDS-PAGE analysis demonstrated dramatic differences in the resolution of the highest-order multimers and large, disordered aggregates of GL-2045 when eluted with 35%, 36%, or 37% EB, or when eluted with elution buffers with higher concentrations of buffer b. Thus, it is clear that selected elution buffers for ion exchange can be used to denature the multimer profile of GL-2045, a novel use of this technology. It is also clear that data from this stepwise elution can be used by one skilled in the art to select a single step or multiple stepwise elutions to obtain a desired GL-2045 profile. For example, the GL-2045 profile using a POROS CIEX column and an elution buffer containing 36.5-38.5% Buffer B was Polish will retain all homodimers, dimers, and multimers through multimer 10 and elute large disordered aggregates and highest order multimers. A similar example using a different column is shown in Example 4.

[0193] Example 13 - Ion exchange chromatography can be used to reduce or remove large homodimer aggregates and highest order multimers Production of an optimal composition of GL-2045 requires removal of material above 1000 kD to minimize the amount of highest-order GL-2045 multimers (e.g., bands above clearly delineable band 10, approximately 600 kD) and remove both large homodimeric aggregates and highest-order multimers, where increasing valency poses a theoretical increased risk of immunogenicity. GL-2045 was eluted with elution buffers containing different percentages of buffer b: 38% EB (C1), 39% EB (C2), and 40% EB (C3) (Figure 26). Major elution peaks for GL-2045 were observed at 38% (C1), 39% (C2), and 40% (C3) (Figure 26), followed by smaller elution peaks at 50% and 100% buffer B. The percent recovery of GL-2045 for each elution buffer was determined and is presented in Table 14. [Table 14]

[0194] The multimer profile of the eluted GL-2045 fractions was determined by visual inspection of SDS-PAGE analysis (Figure 27). Visual inspection of SDS-PAGE analysis of the eluted fractions showed that 38% of the elution buffer recovered GL-2045 with minimal amounts of residual high molecular weight material.

[0195] The GL-2045 peaks were quantified by densitometry (summarized in Table 15 as the percentage intensity of the SDS-PAGE bands, Figure 28). Peak 11 represents the homodimer with the lowest molecular weight, and peak 1 represents the material that preferably contains the highest molecular weight material to be removed. [Table 15]

[0196] These results demonstrate that a stepwise elution protocol using acetate elution buffers containing 38% or 39% Buffer B at pH 5 yielded approximately 85% of the desired fraction of GL-2045 and reduced the higher molecular weight fractions above 600 kD. Visual inspection of the SDS-PAGE indicated that high molecular weight material was lowest in fractions eluted with elution buffer containing 38% Buffer B, while densitometric analysis indicated that elution buffer containing 39% Buffer B yielded the lowest percentage of the highest molecular weight fraction. However, densitometric analysis demonstrated that all of the CIEX-purified protein compositions contained less of the high molecular weight fraction (band 1) compared to material purified by affinity chromatography alone. Overall, elution analysis suggests that the amount of large aggregates and highest-order multimers can be controlled by applying controlled elution conditions using a POROS CIEX column. It is therefore clear that the elution buffer selected for ion exchange can be used to denature large aggregates and maximally highly ordered multimer compositions of GL-2045, a novel use of this technology. A similar example using a different column is given in Example 4.

[0197] Example 14 - Denaturation of GL-2045 multimeric compositions using a hydrophobic interaction chromatography column GL-2045 was generated from a stable HEK 293F cell line and grown in 293-free medium (Gibco #12338-018) with Glutamax (Gibco #35050-061) and Geneticin (Gibco #10131-027). Supernatants were harvested twice weekly and filtered through a 0.2 μm 1 L filter system (Corning #431098). GL-2045 supernatants were then purified using Protein A HiTrap MabSelect SuRe (GE#11-0034-95) with a binding buffer of 20 mM sodium phosphate, pH 7.2, 0.15 M NaCl, and eluted with a 0.1 M sodium citrate elution buffer with a pH of 3.0-3.6. AC-purified GL-2045 was stored in 1X PBS (Quality Biological, Inc. #119-069-101) at pH 7.0.

[0198] GL-2045 was then purified on seven different hydrophobic interaction columns (HIC) using the HiTrap HIC selection kit (GE #28-4110-07). The columns included in this kit are listed in Table 16. [Table 16]

[0199] HIC columns were equilibrated with 50 mM sodium phosphate, pH 7.0, 1.0 M ammonium sulfate (starting buffer), and 3.5 mg of AC-purified GL-2045 (diluted in 4 volumes of starting buffer) was loaded onto each column. After washing with starting buffer, gradient elution was performed using 0% to 100% 50 mM sodium phosphate, pH 7.0. All fraction peaks and flow-through were examined by SDS-PAGE. Unreduced samples were loaded in 15% Tris HCl (Bio-Rad #161-115). Staining was performed using a silver staining kit format, Invitrogen #LC6100.

[0200] Seven different HIC columns demonstrated different multimer profiles for GL-2045. As an example, the Butyl HP column separated the homodimer fraction (fraction A10) from the multimer molecular weight species found in A12. The same effect was seen with the Phenyl HP column (fraction B11 compared to fraction B8). Only 16% of the loaded material was recovered in the elution fractions from the Octyl FF column, indicating that it was a flow-through mode for GL-2045. Polish Furthermore, the fractions eluted from the Octyl FF column contained higher molecular weight species, indicating that the column may also be well suited for removal of very high molecular weight homodimeric aggregate species with lower efficacy.

[0201] A similar experiment was performed on the murine version of GL-2045, known as M045. M045 was purified by Protein A affinity chromatography and then further purified by HIC with Hiload 26 / 10 Phenyl Sepharose High Performance (GE 17-1086-01) on an AKTA Avant (GE). The HIC column was equilibrated with 0.1 M sodium phosphate, pH 7.0, 1 M ammonium sulfate (starting buffer), and M045 was loaded onto the column, followed by a wash step with starting buffer to remove all unbound material. M045 was then eluted with 0.1 M sodium phosphate, pH 7.0 elution buffer by gradient elution (Figures 30-31).

[0202] HIC purified fractions of M045 were analyzed by SDS-PAGE to determine the effect of the HIC column on the M045 multimer profile. Polish The effect of denaturing M045 on the multimeric composition of M045 was determined. These results further demonstrated that a hydrophobic interaction column can be used to denature the multimeric composition of M045, as noted by the clear separation of homodimer, dimer, trimer, and multimer fractions (Figure 32).

[0203] Example 15 - Exemplary Protocol for Optimally Produced GL-2045 The data described herein demonstrate optimal conditions for several variables in the upstream and downstream manufacturing processes of GL-2045 that result in optimization of (1) protein titer, (2) cell viability throughout culture, and (3) GL-2045 multimerization, as well as (4) maintenance of the multimer profile in the final GL-2045 drug substance. Importantly, the level of GL-2045 multimerization is essential for the clinical efficacy of stradomers (see Examples 1-4). Current culture methods do not necessarily aim for optimized production of specific fractions or enhancement of specific multimerization patterns. Thus, the upstream culture reagents and conditions and downstream purification media and conditions that affect multimerization are not all known and cannot be predicted based on the current state of the art.

[0204] The data described herein led to the discovery of the following protocol elements for generating optimally produced GL-2045:

[0205] (1) The optimal base medium for generating optimally produced GL-2045 is ActiCHO P. The data presented herein demonstrate that the optimal basal medium was ActiCHO P. CHO cells cultured in bioreactors in ActiCHO P basal medium were optimized for increased protein titer compared to other basal media tested, while also resulting in high cell density and cell viability. Surprisingly, ActiCHO P medium resulted in an increased percentage of higher order multimers of GL-2045 present at the end of the culture protocol.

[0206] (2) The optimal feeding for generating optimally produced GL-2045 is ActiCHO P and ActiCHO Diet A and Diet B. The data described herein further demonstrate that the optimal feed was ActiCHO P Diet A and Diet B, added to the cultures daily or every other day. ActiCHO P Diet A and Diet B maintained high cell density and viability while producing protein titers four times greater than the other medium / feed combinations tested. Importantly and unexpectedly, ActiCHO P medium with Diet A and Diet B produced high levels of highly ordered multimers and, importantly, a reduced percentage of high molecular weight, disordered aggregates of GL-2045 compared to the other medium / feed combinations tested. These data indicate that this particular medium / feed combination results in the production of a greater percentage of GL-2045 multimers (e.g., a greater percentage of highly ordered GL-2045 multimers) with improved clinical efficacy. The inventors surprisingly found similar results for the addition of Diet A and Diet B every other day, indicating that this particular medium / feed combination can be used to reduce costs and the risk of contamination associated with daily culture manipulations.

[0207] Furthermore, ActiCHO P medium with Diet A and Diet B resulted in the production of a substantial percentage of GL-2045 present as higher-order multimers while minimizing the percentage of unordered, high molecular weight aggregates of GL-2045. Thus, this particular medium / feed combination surprisingly optimized for a biologically functional and clinically effective fraction of highly ordered, multimerized GL-2045, thus optimizing retention of the GL-2045 multimer profile while reducing the need to remove higher-order aggregates in further downstream purification steps.

[0208] (3) The optimal temperature shift for generating optimally produced GL-2045 is a shift of 37 °C to 32.5 °C based on cell density. The data described herein additionally demonstrate that a temperature shift from 37°C to 32.5°C based on cell density results in optimal cell density, viability, and protein titer. This temperature shift protocol deviates from established protocols (Ouguchi et al., Cytotechnology, 52(3), pp. 199-207, (2006); Masterson and Smales, Pharmaceutical Bioprocessing, 2(1), pp. 49-61, (2014)) that describe a temperature shift from 37°C to 31°C based solely on culture days. The inventors unexpectedly found that cells were cultured at approximately 10-15 x 10 6 We found that a temperature shift to 32.5 °C after reaching a density of 100 cells / mL not only maintained high cell density and cell viability, but also resulted in a substantial increase in protein titer compared to previously established protocols.

[0209] The data demonstrated herein demonstrate that specific downstream purification protocols result in GL-2045 compositions with optimized multimerization profiles. Strict care must be taken in implementing these purification methods to maintain the desired multimerization profile of GL-2045 by controlling column conditions and buffers. This is in stark contrast to monoclonal antibodies, Fc fusion proteins, or similar CHO-derived proteins, where purity and retention of yield are primary goals.

[0210] (4) The optimized protein A purification of GL-2045 requires frequent and rigorous CIP procedures. GL-2045 binds avidly to Protein A. This avid binding resulted in GL-2045 remaining bound to the Protein A medium in the column when the CIP procedure commonly used for mAb purification was used. As a result, upon repeated cycles of Protein A column use, the homodimeric fraction of GL-2045 was unable to bind to Protein A and flow through the column. This resulted in substantial and functionally significant changes in the multimer profile of the final Protein A-purified GL-2045 product. Thus, the avid binding of GL-2045 resulted in the requirement for more frequent and stringent CIP procedures (e.g., using a 0.5 M NaOH wash buffer) than those commonly used in the art (e.g., during mAb or Fc-fusion protein purification). These results were unexpected because most commonly used Protein A columns cannot withstand the rigorous NaOH washes required to remove GL-2045 multimers and fully regenerate the Protein A column. Therefore, only partial Protein A columns, specifically MabSelect SuRe, can be used for purification of GL-2045 and will require frequent CIP steps with approximately 0.5 M NaOH to maintain the desired GL-2045 multimer profile.

[0211] (5) A pH elution gradient or step elution facilitates the separation of the highest molecular weight fraction of GL-2045 from lower and higher order multimers. The use of a pH elution gradient with the purification of Protein A resulted in the highest molecular weight components eluting in the first and last elution fractions. These data demonstrate that a pH gradient can be used to separate biologically active fractions of GL-2045 (e.g., homodimers, dimers, and higher multimers) from fractions composed of disorganized, high molecular weight aggregates that were previously shown to have reduced biological activity.

[0212] (6) Ion exchange chromatography of GL-2045 PolishThe optimal elution buffer is acetate buffer + 30-40% buffer B, especially 37.5%-39% + / - 0.5%. Ion exchange chromatography is commonly used to remove impurity drugs during mAb production. Polish However, we used ion exchange chromatography to remove specific fractions of GL-2045 (e.g., the highest order homodimer multimers and high molecular weight disordered aggregates) so that an optimal multimerization profile could be achieved. Specifically, we found that an elution buffer of 30-40% Buffer B reduced the amount of high molecular weight disordered aggregates of GL-2045. Even more specifically, an elution buffer of 38-39% Buffer B reduced the amount of high molecular weight disordered aggregates of GL-2045. The elution buffer was specifically optimized to maintain the amount of homodimer present in the final GL-2045 product while also reducing the amount of disordered aggregates.

[0213] (7) A hydrophobic interaction column (HIC) can be used to achieve a specific GL-2045 multimerization profile. The data herein show that GL-2045 Polish This demonstrates that multiple HIC columns can be used in a process. For example, the flow-through from the Octyl FF column contains primarily high molecular weight species, indicating that this column can be used specifically for the removal of high molecular weight aggregates of GL-2045. Alternatively, a Butyl HP column can be used to separate the homodimer fraction from the multimer fraction for applications where one of the fractions may achieve more desirable results. Alternatively, an HIC column can be used in a combined mode.

[0214] Optimized Manufacturing Protocol for GL-2045 In summary, incorporating all of the parameters discussed above, the following protocol resulted in the highest protein yield of GL-2045 while maintaining the highest percentage of the overall population as multimers.

[0215] CHO cells were transfected with two vectors using ExcellGene SA (Monthey, Switzerland): a GL-2045 expression vector containing a GL-2045 expression cassette flanked by piggyBac transposase target sequences, and a second vector containing piggyBac transposase. The PiggyBac transposon has preferential insertion into highly transcribed regions of the genome and contains inverted terminal repeats, which provide insulation through gene silencing. Transfection resulted in integration of the expression cassette into highly transcribed genomic regions, thereby establishing a bank of stably transfected CHO cells with genomic insertions of less than 20 transgenes. The stably transfected CHO cells were then cultured in a bioreactor in ActiCHO P medium at a growth temperature of 37°C. During this culture, the cells were fed daily with ActiCHO Diet A and Diet B at a growth temperature of 37°C until the culture reached a cell density of approximately 10 to 15 million cells / mL. After reaching such a density, the growth temperature was shifted from 37°C ± 1°C to 32.5°C ± 1°C, and optimally produced GL-2045 was harvested from the medium on the final day of culture.

[0216] This protocol resulted in cell viabilities of greater than 95% on day 18 and greater than 80% on day 21 of culture, and a final total protein titer of greater than 9,000 mg / mL, with greater than 70% of the GL-2045 present as non-homodimers and greater than 30% present as higher order multimers above the fifth multimer.

[0217] GL-2045 was harvested from the culture supernatant using a tangential flow filtration system, which did not interfere with the passage of the most highly ordered multimers and thereby preserved the homodimer and multimer profile of the supernatant. Downstream production methods were then used to isolate GL-2045, remove impurities, and isolate specific fractions to control the multimer profile of GL-2045 (e.g., remove unordered, high-molecular-weight aggregates). GL-2045 was purified by protein A affinity chromatography, and protein A medium was selected for its ability to withstand highly alkaline renaturation. Furthermore, more than one wash buffer was used to allow for further control over the purification process. Additionally, a CIP procedure was performed more frequently than usual to fully regenerate the binding capacity of the protein A column, as required to retain homodimers in the final GL-2045 composition, and a 0.5 M NaOH buffer was used to remove GL-2045 multimers that had bound to the column. GL-2045 was eluted from the Protein A column with or without a pH elution gradient. After purification by fluoroscopy, additional Polish The process was used. Cation exchange chromatography was used to remove high molecular weight disordered aggregates of GL-2045 with an elution buffer containing 37-39% + / - 0.5% buffer b, preferably with CIEX POROS XS resin. In some embodiments, GL-2045 was further purified using an HIC column. To remove high molecular weight disordered aggregates of GL-2045, Octyl FF resin was added with additional Polish Alternatively, a Butyl HP-containing column was used to isolate specific GL-2045 fractions (e.g., to isolate higher order multimers). In addition, an anion exchange column, specifically a Q Sepharose fast flow column, was used as an additional step, especially in flow-through mode. Polish was used as a process.

[0218] Although these additional purification steps can be used individually, purification of GL-2045 by Protein A affinity chromatography was preferably used in combination with all three of anion exchange, cation exchange, and hydrophobic interaction chromatography to arrive at a final GL-2045 drug substance with a final protein titer of >4 g / L, >70% of the GL-2045 present as multimers, and >30% of the multimers being pentamer or higher multimers.

[0219] Example 16 - Analysis of optimally produced GL-2045 To further characterize the optimally purified GL-2045 produced by the methods described herein, GL-2045 was produced in a bioreactor according to the upstream methods described herein using the ActiPro basal medium, feed, and temperature shift described herein. The resulting GL-2045 supernatant was then passed through a Millipore X0HC depth filter, followed by filtration through a 0.2 μm filter and processed using multiple downstream processing methods. The multimerization profile of the GL-2045 composition was assessed after each processing step and is shown in Figure 33. The multimerization profile of the filtered GL-2045 preparation is shown by the red dots in Figure 33. The filtered GL-2045 preparation was then subjected to affinity chromatography with Protein A MabSelect Sure (GE.#11-0034-95) (multimerization profile shown by the blue dots in Figure 33) and then purified using AIEX in flow-through mode using a Q Sepharose fast flow column. The resulting GL-2045 was then pH adjusted to pH 5.0±0.10 and filtered through a 0.2 μm filter (multimerization profile shown by green dots in Figure 33). GL-2045 was then purified by cation exchange chromatography using a Poros XS column in binding mode by step elution using an elution buffer containing 50 mM Na acetate + 375 mM NaCl at pH 5.0 (multimerization profile shown by yellow dots in Figure 33), followed by hydrophobic interaction chromatography (multimerization profile shown by orange dots in Figure 33), and filtration (multimerization profile shown by purple dots in Figure 33) to arrive at the final drug substance (multimerization profile shown as black dots in Figure 33). The raw data for Figure 33 is shown below in Table 17. [Table 17-1] [Table 17-2] [Table 17-3] [Table 17-4]

[0220] After each analytical HPLC step, the percentages of homodimer, dimer, trimer, tetramer, pentamer, hexamer, and 7+mer fractions were assessed. Briefly, the supernatant of stably transfected CHO cells of GL-2045 was generated according to the upstream method described herein. GL-2045 was then purified according to the downstream method described herein. Samples were obtained at the following successful purification steps: Protein A MabSelect SuRe load, Protein A MabSelect SuRe pool, anion exchange pool, cation exchange pool, HIC pool, UFDF pool, and drug substance. The samples were compared by analytical SEC-HPLC. Briefly, isocratic separation was performed by HPLC on a high-performance liquid chromatography system (Agilent 1100 HPLC system) using a series of two SEC columns (Agilent Bio SEC (300 Å)) with UV detection at 280 nm. Chromatography is performed with a run time of 60 minutes and a flow rate of 0.5 mL / min. The relative area percentage of each peak is calculated.

[0221] The results are shown in Figure 33. As is evident in Figure 33, downstream processing of GL-2045 altered the smallest fractions, the homodimer and homodimer dimer, and the largest fraction, the 7mer+, while fractions 3-6 remained fairly stable. For the smaller multimers, progressive downstream manufacturing steps from loading the Protein A column to the final drug substance resulted in increased recovery of the homodimer and homodimer dimer. However, progressive downstream processing steps had the opposite effect on the highest order multimers, resulting in a decrease in their associated percentages.

[0222] The resulting GL-2045 drug product had a defined multimer pattern, comprising less than about 20% homodimers and more than about 28% 7-mers or greater, as a percentage of the total composition. The composition also contained about 7-12% homodimer dimers, about 6-11% homodimer trimers, about 10-16% homodimer tetramers, about 6-9% homodimer pentamers, and about 10-14% homodimer hexamers.

Claims

1. 1. A composition comprising a recombinantly produced homodimer, wherein the homodimer comprises two monomers each comprising amino acids 21-264 of SEQ ID NO:4, wherein the homodimer comprises less than 20% of the total composition, and wherein multimers of the homodimer comprise more than 80% of the total composition.

2. 1. A composition comprising a recombinantly produced homodimer and a multimer of said homodimer, the homodimer comprises two monomers each comprising amino acids 21-264 of SEQ ID NO:4; The homodimers constitute less than 20% of the total composition, and the multimers of the homodimers are (a) pentamers or greater of said homodimers comprising at least about 28% of said total composition; (b) a dimer of said homodimer comprising from about 7% to about 13% of said total composition; (c) a trimer of said homodimer, comprising from about 5.5% to about 11% of the total composition; (d) a tetramer of said homodimer, comprising 10% to about 16% of the total composition; (e) a pentamer of said homodimer, comprising from about 6% to about 10% of said total composition; (f) hexamers of said homodimers, comprising from about 10% to about 14% of the total composition; (g) the dimer of the homodimer through the hexamer of the homodimer, comprising from about 39% to about 61% of the total composition; (h) the trimer of the homodimer through the hexamer of the homodimer, comprising from about 32% to about 50% of the total composition; (i) the tetramer of the homodimer through the hexamer of the homodimer, comprising from about 26% to about 39% of the total composition; (j) the pentamer of the homodimer through the hexamer of the homodimer comprising from about 16% to about 23% of the total composition; or (k) a composition comprising any combination of (a) through (j).

3. 1. A composition comprising a recombinantly produced homodimer and a multimer of said homodimer, the homodimer comprises two monomers each comprising amino acids 21-264 of SEQ ID NO:4; The homodimers constitute less than 20% of the total composition, and the multimers of the homodimers are (a) pentamers or greater of said homodimers comprising at least about 28% of said total composition; (b) a dimer of said homodimer comprising from about 7% to about 13% of said total composition; (c) a trimer of said homodimer, comprising from about 5.5% to about 11% of the total composition; (d) a tetramer of said homodimer, comprising 10% to about 16% of the total composition; (e) a pentamer of said homodimer comprising from about 6% to about 10% of said total composition; and (f) a hexamer of said homodimer comprising about 10% to about 14% of the total composition.

4. 1. A composition comprising a recombinantly produced homodimer and a multimer of said homodimer, the homodimer comprises two monomers each comprising amino acids 21-264 of SEQ ID NO:4; The homodimers constitute less than 20% of the total composition, and the multimers of the homodimers are (a) pentamers or greater of said homodimers comprising at least about 28% of said total composition; (b) the dimer of the homodimer through the hexamer of the homodimer, comprising from about 39% to about 61% of the total composition; (c) the trimer of the homodimer through the hexamer of the homodimer, comprising from about 32% to about 50% of the total composition; (d) the tetramer of the homodimer through the hexamer of the homodimer, comprising from about 26% to about 39% of the total composition; and (e) a composition comprising from about 16% to about 23% of the total composition of the pentamer of the homodimer to the hexamer of the homodimer.

5. A pharmaceutical composition for treating or preventing an inflammatory, autoimmune, or infectious disease or disorder in a subject in need thereof, comprising the composition of any one of claims 2 to 4.

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