Manufacturing optimization of the polymerized stradomer GL-2045

Abstract

Providing manufacturing optimization for the multimerized stradomer GL-2045. [Solution] A method for producing a composition containing a homodimer and / or a multimer of the homodimer, wherein the homodimer contains two monomers, each monomer containing amino acids 21 to 264 of a specific sequence, the method comprising: (a) culturing Chinese hamster ovary (CHO) cells stably transfected with an expression vector containing a nucleic acid sequence encoding the specific sequence at a growth temperature of 37°C±1°C for 5±1 days; (b) shifting the growth temperature from 37°C±1°C to 32.5°C±1°C; and (c) collecting the homodimer and / or the multimer of the homodimer from the culture medium.

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 Electronically Submitted Text Files The contents of the text files submitted electronically herein are incorporated by reference in their entirety. Computer Readable Format Copy of Sequence Listing (Filename: GLIK_017_01WO_ST25.txt; Date of Record: December 8, 2017; File Size, 15 kb).

[0003] The present invention generally relates to the fields of immunology, autoimmunity, inflammation, and tumor immunology. More specifically, the present invention relates to an optimized method for producing GL-2045. The present invention also relates to novel compositions comprising such optimized produced GL-2045, and methods of 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 immune deficiencies since the early 1950s and autoimmune and inflammatory diseases in the following decades. 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 that typically contains greater than 90% native IgG with small and variable amounts of the polymeric immunoglobulins, IgA or IgM (Rutter A et al., J Am Acad Dermatol, 2001, June;44(6):1010-1024).

[0005] Substantial published data suggests 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 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 (US Patent Application Publication No. 2013 / 0156765). GL-2045 is a multimerizing generic stradomer that is a recombinant mimetic of IVIG. GL-2045 binds most or all of the ligands that immunoglobulin IgG1 Fc binds. Furthermore, GL-2045 binds with high affinity and avidity to all standard receptors and to complement C1q, and has 10-1,000-fold greater in vitro potency compared to IVIG. In addition, 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. Thus, 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 that it is obtained from the blood of tens of thousands of donors, and then their serum is mixed together and subsequently purified to remove viruses and other infectious agents, as well as aggregated IgG. As such, it is limited in availability and supply, and expensive to produce. In addition, there is a significant degree of variability between large quantities of IVIG. Conversely, GL-2045 is recombinantly produced, which eliminates the difficulties of supply and production costs, 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 the multimer fraction of IVIG.However, standard cell culture conditions produce variable levels of cell viability, degree of multimerized protein, and protein titer.Therefore, there is a need in the art for a method of producing GL-2045 that produces a defined multimer pattern, and in particular, 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 Derm atol,2001,June;44(6):1010-1024 Summary of the Invention [Means for solving the problem]

[0011] The present invention provides all three of the above, improved cell viability, improved high protein titer, and a surprising substantial increase in the percentage of higher order multimers over standard manufacturing techniques. 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 methods. Optimized upstream manufacturing methods a) generate high protein titers, b) maintain high cell viability and minimize cell debris, and c) retain both homodimer highly ordered multimers, which are essential for the functionality of GL-2045, and homodimers, as desired. Optimized downstream manufacturing methods include various purification techniques that are specifically used to maintain a selected multimer profile of GL-2045. Thus, in some embodiments, GL-2045 compositions with defined multimer profiles are provided herein.

[0012] In some embodiments, a method for producing GL-2045 is provided, 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 prior to the shift in growth temperature. In some embodiments, the cells are grown to a density of about 10 million to about 15 million cells / mL prior to the shift in growth temperature. In some embodiments, the cells are grown to a density of about 15 million to about 20 million cells / mL prior to the shift in growth temperature. In some embodiments, a dual temperature shift is used, with a shift from 37°C ± 1°C to 34°C ± 1°C on about day 3 of the 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 Feed A and ActiCHO Feed B during culture. In some embodiments, the CHO cells are fed every other day. In some embodiments, the expression vector encoding GL-2045 comprises a 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, there is provided a recombinantly produced GL-2045 made by the methods described herein. In some embodiments, there is provided 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 with 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 results in >80% cell viability at day 21 and a final protein titer of >9,000 mg / mL, of which >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, methods are provided for purifying GL-2045 produced by the methods described herein, comprising purifying the GL-2045 from the culture supernatant by affinity chromatography and polishing the GL-2045 by one or more of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography.

[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 Emphaze™ AEX Hybrid Purifier (3M).

[0018] In some embodiments, the 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 a MabSelect SuRe resin. In some embodiments, the affinity chromatography purification comprises optimizing purification conditions utilizing one of three different wash buffers. In some embodiments, the affinity chromatography purification comprises eluting GL-2045 from the affinity chromatography column. In some embodiments, the elution of GL-2045 comprises elution with a pH gradient. In some embodiments, the elution of GL-2045 comprises elution without a pH gradient. In one embodiment, the elution is performed using a glycine buffer. In another embodiment, the 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, the polishing of GL-2045 includes an anion exchange flow through chromatography. In some embodiments, the anion exchange flow through chromatography includes using a Q Sepharose fast flow column. In some embodiments, the polishing of GL-2045 includes cation exchange chromatography. In some embodiments, the cation exchange chromatography includes using a POROS XS column. In some embodiments, the cation exchange chromatography includes using a sodium acetate elution buffer. In some embodiments, the elution buffer further includes 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 polishing of GL-2045 includes hydrophobic interaction chromatography. In some embodiments, the hydrophobic interaction chromatography includes using a Butyl FF resin. In some embodiments, the hydrophobic interaction chromatography includes using a Phenyl HP resin. In some embodiments, hydrophobic interaction chromatography ("HIC") involves using a Phenyl Sepharose 6 fast flow high sub-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 involves 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, where 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 is provided, comprising polishing the GL-2045 by cation exchange chromatography, wherein the cation exchange column contains a high capacity, high resolution resin, such as POROS XS, and the elution buffer is a sodium acetate buffer composed of 36.5-39.0% 1 M NaCl buffer. In some embodiments, the method for purifying GL-2045 further comprises polishing the GL-2045 by anion exchange 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. In some embodiments, the method for purifying GL-2045 further comprises polishing the GL-2045 by hydrophobic interaction chromatography, where the hydrophobic interaction medium is Butyl FF, Phenyl HP, or Octyl FF resin, and in addition to polishing, is selected to isolate or remove a specific fraction of GL-2045. In some embodiments, the method for purifying and / or polishing GL-2045 results in a final protein titer of >4g / L of GL-2045 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, 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, a method is provided for treating or preventing an inflammatory, autoimmune, or infectious disease or disorder in a subject in need of treatment or prevention using recombinantly produced purified GL-2045 as 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, bucally, 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 that comprises 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, a recombinantly produced GL-2045 composition is provided, wherein the highest order multimers (i.e., in the homodimer 7-mer or greater fraction) 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 constitutes less than about 20% of the total composition; (b) the highest 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 constitutes about 5.5% to about 11% of the total composition; (e) the homodimer tetramer fraction constitutes about 10% to about 16% of the total composition; (f) the homodimer pentamer fraction constitutes 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 constitutes 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)-(k).

[0031] In some embodiments, a recombinantly produced GL-2045 composition is provided in which approximately 80% of the total composition comprises higher order multimers, meaning dimers or more of homodimers (i.e., 2 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., 3 or more bands). In some embodiments, approximately 54-72% of the total recombinantly produced GL-2045 composition comprises tetramers or more (i.e., 4 or more bands). In some embodiments, a recombinantly produced GL-2045 composition is provided in which approximately 44-57% of the total composition comprises pentamers or more (i.e., 5 or more bands). In some embodiments, approximately 38-51% of the total recombinantly produced GL-2045 composition comprises hexamers or more (i.e., 6 or more bands).

[0032] In some embodiments, recombinantly produced GL-2045 is provided, in which 2-6 bands of the composition (i.e., homodimer dimer to homodimer hexamer) constitute about 39-61% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, in which 3-6 bands of the composition (i.e., homodimer trimer to homodimer hexamer) constitute about 32-50% of the composition. In some embodiments, recombinantly produced GL-2045 is provided, in which 4-6 bands of the composition (i.e., homodimer tetramer to homodimer hexamer) constitute about 26-39% 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) constitute about 16-23% of the composition. [Brief description of the drawings]

[0033] [Figure 1A]Illustrated is 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 is GL-2045 fractionation by size exclusion chromatography (FIG. 1A) and analysis of the resulting fractions on a non-reducing gel (FIG. 1B). [Diagram 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] Illustrated are the binding of the eluted fractions shown in FIG. 5 to FcγRIIIa (FIG. 6A) and the best-fit curves (FIG. 6B). [Figure 6B]Illustrated are the binding of the eluted fractions shown in FIG. 5 to FcγRIIIa (FIG. 6A) and the best-fit curves (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 = 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 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] Illustrates protein titers (mg / mL) of CHO cells grown in a panel of different media on day 10 of culture. [Figure 12A] FIG. 12 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] FIG. 12 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]Illustrated are feeding schedules for PowerCHO3 CD, ADCF-Mab Hyclone, and ActiCHO P media (FIG. 13A), and feeding schedules for Cellvento, BalanCD CHO Growth A, CD FortiCHO Life, and CD4MCHO Hyclone media (FIG. 13B). [Figure 13B] Illustrated are feeding schedules for PowerCHO3 CD, ADCF-Mab Hyclone, and ActiCHO P media (FIG. 13A), and feeding schedules for Cellvento, BalanCD CHO Growth A, CD FortiCHO Life, and CD4MCHO Hyclone media (FIG. 13B). [Figure 14] Illustrates 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 % 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] FIG. 13 illustrates an 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 (Figure 18A), cell viability (Figure 18B), culture pH (Figure 18C), and GL-2045 protein titer (Figure 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 (Figure 18A), cell viability (Figure 18B), culture pH (Figure 18C), and GL-2045 protein titer (Figure 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 (Figure 18A), cell viability (Figure 18B), culture pH (Figure 18C), and GL-2045 protein titer (Figure 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 (Figure 18A), cell viability (Figure 18B), culture pH (Figure 18C), and GL-2045 protein titer (Figure 18D). [Figure 19] Illustrates the effect of daily ActiCHO-P medium + feeding (red) and every 3 days ActiCHO-P medium + 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] Illustrates SDS-PAGE analysis of Protein A purified GL-2045. [Figure 23A] Illustrated is the elution profile from a Protein A column following 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 following 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. [Diagram 25] Elution chromatograms and non-reducing SDS-PAGE analysis are illustrated. [Figure 26] The elution chromatographs of runs C1 to C3 are illustrated. [Figure 27] Illustrated is a non-reducing SDS-PAGE analysis of elution peaks 38%-39% from runs C1-C3. [Figure 28] Illustrates densitometric analysis of ion chromatography purified GL-2045. [Figure 29A] Illustrated is the elution profile of the HIC column (upper panel) and SDS-PAGE analysis of the eluted and flow-through fractions (FT) (lower panel). [Figure 29B] Illustrated is the elution profile of the HIC column (upper panel) and SDS-PAGE analysis of the eluted and flow-through fractions (FT) (lower panel). [Diagram 30] Illustrates the elution profile from HIC column polishing (right panel) and Nu-PAGE analysis (right panel) of M045. [Diagram 31] 1 illustrates the elution profile from HIC column polishing of M045. [Diagram 32] Illustrates the elution profile and SDS-PAGE analysis of M045 from HIC column polishing. [Diagram 33] 1 illustrates the defined multimer pattern of an optimally manufactured GL-2045 composition. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0034] The approaches to the generation of 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 that include GL-2045 with defined multimer 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 words "a" or "an" when used in conjunction with the term "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 an Fc fragment of an antibody. "Biologically active" biomimetics 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 naturally occurring. "Immunologically active" biomimetics are biomimetics 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 multimerized stradomers, as defined herein (e.g., optimally manufactured GL-2045).

[0038] "Directly linked" refers to two sequences that are connected to each other without any intervening or extraneous sequence, such as 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 multimerization ability is not substantially affected.

[0039] "Homologous" refers to the identity over the entire sequence of a given nucleic acid or amino acid sequence. For example, "80% homologous" means that the given sequence shares about 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 over the entire length of the sequence.

[0040] It has been described that hIVIG binds to and completely saturates the neonatal Fc receptor (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 bind strongly 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 include either the DEL polymorph or the EEM IgG1 polymorph. Thus, even if 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 results.

[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 stradomers 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 binding 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 contains at least one Fc domain and, in the case of GL-2045 and an IgG2 hinge multimerization domain, forms a homodimeric "stradomer unit" when associated with at least a second stradomer unit monomer. In a preferred embodiment, the stradomer unit of GL-2045 is composed of two associated stradomer unit monomers. However, a GL-2045 stradomers may also contain more than two stradomer unit monomers.

[0044] The optimally engineered stradomer of the 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, one of skill in the art will understand that an optimally manufactured stradomer molecule of the invention may be constructed by preparing a polynucleotide molecule encoding an Fc domain monomer and a multimerization region. Such a polynucleotide molecule may be inserted into an expression vector, which may be used to transform a population of bacteria or transfect a population of mammalian cells. Stradomer unit monomers may then be produced by culturing the transformed bacteria or transfected mammalian cells under appropriate culture conditions. For example, by selecting the cells with geneticin / G418, a clonal cell line may be achieved that continues the pool of stably transfected cells. Alternatively, cells may be transiently transfected with DNA encoding an optimally manufactured stradomer of the 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 may then form functional stradomer units and stradomers upon either self-assembly of stradomer monomers or units or association of stradomer monomers using the interconnection 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 only the production of the stradomer unit monomer peptides 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 Stradomer In one embodiment, the optimally manufactured GL-2045 made according to the present disclosure is a cluster stradomer. A "cluster stradomer" is a biomimetic with a radial morphology that includes a central "head" and two or more "legs," each of which includes one or more Fc domains capable of binding at least one Fc gamma receptor and / or complement. Cluster stradomers are also known as "multimerized stradomers" due to the presence of multimerization domains that result in multimerization of the stradomer. Thus, serial stradomers that contain multiple Fc domains on one stradomer monomer molecule can still be classified as cluster stradomers or multimerized stradomers, as long as the molecule also contains at least one multimerization domain. Each cluster stradomer is composed of more than one homodimeric protein, each of which is 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 "foot" region that includes at least one functional Fc domain. The multimerization region, once multimerized to another cluster stradomer unit, creates the "head" of the cluster stradomer. The foot region may be capable of binding as many complement molecules as there are Fc domains in each foot region. For example, the foot region may be capable of binding as many C1q molecules as there are Fc domains in each foot region. Thus, cluster stradomers are biomimetic compounds that can bind more than one C1q molecule, thus 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 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 CC 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 homodimer dimers is less than 10% of 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 manufactured 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 3-month stability studies at 37°C and 100% humidity. Furthermore, it is surprising that the amount of multimers in IgG2 hinge-containing stradomers formulated drugs is significantly greater than the approximately 10% of dimers observed for IgG2 in human serum, and does not include multimers. For example, the percentage 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 percentage of GL-2045 that exists as homodimers is between 10-20% and the corresponding percentage 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 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 proteins and other naturally occurring organic molecules with which it is naturally associated. Preferably, a preparation of the polypeptide (or peptide) of the invention is at least 80%, more preferably at least 90%, and most preferably at least 99% by dry weight of the polypeptide (peptide) of the invention. A synthetic polypeptide or peptide is "isolated" because a chemically synthesized polypeptide or peptide is essentially separated from components that naturally accompany it.

[0050] The 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 system 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, the 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 suitable method, for example, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.

[0051] Manufacturing method GL-2045 forms ordered multimers of homodimers and is active in all of the 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 homodimers and highly ordered multimers of GL-2045 that produces the desired biological outcome for GL-2045 as an IVIG mimetic (e.g., without being bound by theory, at the level of C3 / C3b, for example, initial activation of the complement system, and / or subsequent inhibition of complement activation, and improved binding to C1q by preventing CDC). 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, the larger multimer fractions of GL-2045 are more active than the 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 that include optimized multimer profiles, but also to GL-2045 compositions that only involve selecting multimers based on desired effector functions. In such embodiments, the optimized multimer profile of GL-2045 that produces one desired biological outcome may differ from the optimized multimer profile that produces another desired biological outcome.

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

[0053] In some embodiments, the optimized manufacturing methods described herein result in a GL-2045 protein composition in which at least 70% or at least 80% of the GL-2045 exists as a non-homodimer (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 a non-homodimer. For example, the optimized manufacturing methods may result in a GL-2045 protein composition in which 80%, 85%, 90%, 95% or more of the GL-2045 exists as a non-homodimer. In some embodiments, the protein composition comprises at least 28% or at least 30% of the GL-2045 present as a highest order multimer (i.e., 7-mers or more of homodimers). In some embodiments, the protein composition comprises no more than 35% of the GL-2045 present as a highest order multimer. 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 fractions or higher). 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 fractions or higher). In some embodiments, at least about 35% of 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). In some embodiments, at least about 35% of GL-2045 exists as pentamers or higher of homodimers (i.e., at least 35% of the total GL-2045 composition is composed of 5 fractions or higher).In some embodiments, at least about 35% of GL-2045 exists as homodimer pentamer and hexamer (i.e., at least 35% of the total GL-2045 composition is composed of 5 and 6 fractions). In some embodiments, at least about 35% of GL-2045 exists as homodimer hexamer or higher (i.e., at least 35% of the total GL-2045 composition is composed of 6 fractions or higher). In some embodiments, at least about 35% of GL-2045 exists as homodimer 7-mer or higher (i.e., at least 35% of the total GL-2045 composition is composed of 7 fractions or higher). For example, the optimized manufacturing methods described herein can result in GL-2045 protein compositions in which 40%, 45%, 50%, 55% or more of GL-2045 exists as homodimer pentamer or higher. Current manufacturing methods for Fc-containing therapeutics (e.g., monoclonal antibodies) have focused on increasing protein titer and increasing yield through downstream filtration steps. These methods generally do not address the effect of manufacturing processes on Fc-containing protein multimerization, 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 the manufacturing variables that affect overall protein titer are, at least in part, independent of the variables that affect multimerization profile. Thus, 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 established for the production of recombinant proteins using Chinese Hamster Ovary (CHO) cells provide for a temperature shift from 37°C to 31°C on certain 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 in Ouguchi et al., we have found that a temperature shift from 37°C to 32.5°C results in the maintenance of 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 enhancement of GL-2045 protein titer of approximately 10 g / L.

[0055] Furthermore, the inventors have discovered that the maintenance of an optimized multimerization profile resulting from an optimized upstream manufacturing process is partially dependent on an optimized downstream manufacturing process (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 denaturation 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 to use according to Good Manufacturing Practice (GMP) purification of a single drug, and needs to be reused as many as 100 times or more to be economically viable. Similar to 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 utilization of 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 inventors further discovered that the protein A column clean-in-place (CIP) procedure commonly used in the art unexpectedly results in a change in the GL-2045 multimerization profile. The normal CIP procedure entails column cleaning at the end of a purification run, which may involve multiple cycles of protein supernatant passing through the column. However, the inventors discovered that the high binding strength of GL-2045 results in a lack of dissociation of GL-2045 from protein A. As a result, the binding sites of protein A remain occupied, preventing the binding of GL-2045 in subsequent cycles, resulting in the loss of homodimers. Thus, in contrast to protocols utilized with mAbs or Fc fusion proteins, the inventors unexpectedly discovered that CIP cleaning of protein A columns with GL-2045 must be performed more frequently than with monoclonal antibodies or Fc fusion proteins, and with highly stringent regeneration buffers such as 0.5M NaOH.

[0057] pH elution gradients are commonly used with Protein A columns to optimize protein yield during purification. The inventors have 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 homodimer or higher multimer components of GL-2045, thus altering the multimerization profile of GL-2045 (as demonstrated in Example 10). Thus, the inventors have also determined a means to use elution gradient techniques to optimize the combination of GL-2045 yield and multimerization. In addition, the inventors have surprisingly discovered that pH elution gradients can be applied to Protein A columns for a novel purpose, namely to separate the distinct maximally highly ordered GL-2045 multimers from homodimer aggregates (as demonstrated in Example 11).

[0058] The 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 no change in the composition of the drug. In addition, the inventors have surprisingly discovered that salt and / or pH adjustments can be used to separate the most highly ordered GL-2045 multimers from homodimeric aggregates that may be of similar molecular mass for novel purposes. As demonstrated in Example 13, the 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 commonly used in the art to purify monoclonal antibodies and Fc fusion proteins through a variety of mechanisms, including high yield capture, polishing of monoclonal antibodies, removal of small length species from full length forms, separation of active from inactive forms, and clearing viruses. 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 are associated with widely different capture rates ranging from 16% to 62%, even though the same supernatant and the same buffer are used with all columns.

[0060] Furthermore, we predict that a change in buffer may alter the multimerization profile of GL-2045. This is in stark contrast to monoclonal antibodies or Fc fusion proteins, where a change in buffer may result in a loss of small amounts of protein or a less complete polish, but would not alter the underlying composition of the drug.

[0061] Furthermore, the inventors have discovered that because the GL-2045 homodimer is composed of 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 it binds to without having the avidity to native IgG1 Fc homodimers. 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 less desirable potential target binding, such as endotoxin. For this reason, the inventors have determined that a multi-step purification process is desirable, including purification of GL-2045 with protein A, and polishing of the protein A purified GL-2045 with at least one or more cation exchange chromatography, anion exchange flow-through, and hydrophobic interaction columns. In a preferred embodiment, a four-step purification process is desirable, including purification of GL-2045 by protein A, and polishing the protein A purified GL-2045 by all three of cation exchange chromatography in binding mode, anion exchange in flow-through mode, and hydrophobic interaction column in either binding mode or flow-through mode. This four-step purification process is outlined in Example 15. Those skilled in the art will readily understand that additional filtration steps, including depth filtration and ultrafiltration steps, can be added at any time before, during, or after the process described in Example 15 to further purify the GL-2045 composition.

[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 particular 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. An upstream production method with modifications or changes aimed at optimizing the properties of a particular protein (e.g., efficiency of multimerization) is referred to herein as an "optimized upstream production method." To yield a final protein product with specific properties, some aspects of the upstream protein production may be optimized (e.g., changed or altered to achieve a desired result). Aspects of upstream recombinant protein production that may be optimized may include, but are not limited to, the composition of the expression vector encoding the protein, cell type, basal medium, media additives including feeding, 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 production 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 production methods.

[0063] In some embodiments of the 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. Co-expression 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), which are incorporated herein by reference). The piggyBac system typically increases protein production, at least in part due to the large number of integrated transgenes. However, while the inventors selected high titer, high viability clonal cell lines with relatively low transgene insertion rates (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 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 to be stably transfected once with the GL-2045 expression vector using a technique that causes preferential insertion at transcriptionally active sites. In some embodiments, the culture conditions applied to the transfected selected CHO cell line allow the culture protocol to continue longer than with 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 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 in 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 the 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 quantities as ActiCHO P. ActiCHO P is also currently sold 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 sold 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 prior to the temperature shift. In some aspects, the CHO cells are grown to a density of about 5-30 million cells / mL prior to 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 prior to the temperature shift. In some aspects, the CHO cells are grown to a density of about 10-25 million cells / mL prior to 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 prior to 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 (Ouguchi et al, Cytotechnology, 52(3), pp. 199-207, (2006); Masterson and Smales, Pharmaceutical Bioprocessing, 2(1), pp. 49-61, (2014)), in which cells are often cultured at 37°C for a predetermined number of days and then the temperature is shifted to 31°C. 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 over 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 the third or fourth day 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 5-20 million cells / ml at the first temperature shift. In a particularly preferred embodiment, the cell density is 8-15 million cells / ml at the first temperature shift. In some embodiments, the 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, the harvesting of GL-2045 is accomplished by downstream manufacturing methods. In some embodiments, downstream manufacturing methods are used in combination with the optimized upstream manufacturing 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 manufacturing 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 manufacturing method is optimized for the purification and filtration of GL-2045 to generate and / or maintain a specific multimerization profile of GL-2045, referred to herein as an "optimized downstream manufacturing 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. Reuse of a Protein A column entails "regeneration" of the Protein A column to maintain protein binding capacity. "Regeneration" or "regenerating" as used herein 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 an embodiment, 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 a preferred embodiment, the 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 a NaOH concentration greater than 0.3 M NaOH. For example, the 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 or more NaOH. In a particular embodiment, 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 agent. 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 method of GL-2045 includes a multi-step purification process including purification by affinity chromatography (e.g., protein A affinity chromatography) and at least one or more polishing steps selected from cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction columns. In a preferred embodiment, a four-step purification process for GL-2045 is used instead of the two or three step purification process commonly practiced in the art, including purification by affinity chromatography (e.g., protein A affinity chromatography) and polishing by each of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction columns. The term "polishing" classically refers to the post-protein A purification removal of remaining impurities, including aggregates, endotoxins, DNA, and / or viruses. In addition, with respect to GL-2045, "polishing" also means controlling the proportion of homodimers and certain higher order multimers, such as through the use of these same chromatographic techniques.

[0072] In some embodiments, GL-2045 is polished by ion exchange chromatography (e.g., cation or anion exchange). In some embodiments, polishing of GL-2045 by ion exchange chromatography is performed with an elution buffer that reduces and / or minimizes the amount of disordered high molecular weight aggregates of GL-2045 homodimers that are retained during post-Protein A purification processes. 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 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%-40% buffer B. In some embodiments, the elution buffer comprises 35%-40% buffer B. In still further embodiments, the elution buffer comprises 37%-39% buffer B. In some embodiments, the elution buffer comprises 38%+ / -0.5% buffer b.

[0073] In some embodiments, GL-2045 is polished using hydrophobic interaction chromatography (HIC). In some embodiments, a HIC column (e.g., Octyl FF HIC column) is selected to remove high molecular weight disordered aggregates of GL-2045. Either flow-through or binding mode can be performed to purify GL-2045 using a HIC column. Those skilled in the art will understand that adjusting variables such as pH and salt conditions will determine whether GL-2045 will bind to the HIC resin or flow through the column. In some embodiments, a HIC column is selected to purify specific fractions of GL-2045, such as homodimers and / or higher multimers (e.g., Butyl HP and / or Phenyl HP columns). In some embodiments, it may be desirable to isolate specific fractions of GL-2045 for the treatment of specific disease indications. For example, a HIC column can be used to generate a pharmaceutical substance composed of a particular GL-2045 fraction (e.g., a pharmaceutical substance composed mainly of GL-2045 homodimers, a pharmaceutical substance composed mainly of dimers of homodimers, a pharmaceutical substance composed of higher order multimers of GL-2045, etc.). Separation of the GL-2045 fraction into separate products can be beneficial for certain disease indications. For example, GL-2045 homodimers bind to FcγRI but substantially bind to other FcRs. Thus, GL-2045 homodimers can be particularly useful for treating diseases mediated, at least in part, by FcγRI signaling, such as peritonitis (Heller et al., J.Immunol, V.162, 1992) or acute lung injury (Xie et al. J.Immunol., V 188, 2012). Similarly, trimers, potentially dimers and tetramers of GL-2045 may be particularly useful for treating autoimmune diseases (see WO2015 / 168643). Since 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 some of the ways in which GL-2045 fractions may be useful for treating certain diseases.

[0074] In some embodiments, the optimized downstream manufacturing method described herein may be a combination of individual purification and / or filtration techniques. For example, in some embodiments, the optimized downstream method may include purification of GL-2045 by affinity chromatography (e.g., by an optimized method for a protein A column), followed by additional polishing by an ion exchange chromatography method (e.g., by an optimized cation exchange method), and / or a hydrophobic interaction column. In some embodiments, the optimized downstream method described herein includes 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 manufacturing method" or "optimized manufacturing method," as used interchangeably herein, can refer to optimized upstream and / or optimized downstream manufacturing methods. In some embodiments, optimized manufacturing methods include both optimized upstream and downstream methods. Thus, as used herein, the terms "optimally manufactured stradomer" or "optimally manufactured GL-2045" refer to high titer, high order multimer predominant GL-2045 compositions made according to optimized upstream manufacturing conditions and / or optimized downstream manufacturing methods. The GL-2045 compositions described herein can be optimally produced GL-2045 (i.e., GL-2045 made by the methods described herein), although one of skill in the art will understand that GL-2045 compositions that fall 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 comprising GL-2045, including the GL-2045 drug substance, regardless of whether the composition has been made via an optimized 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 FIG. 33.

[0076] In some embodiments, provided herein are recombinantly produced GL-2045 compositions with defined multimer patterns. As used herein, the term "defined multimer pattern" or "defined multimerization pattern" or "defined band pattern" refers to a pattern of GL-2045 multimerization that is reproducible and can be described in terms of the percentage of the total 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. As an example, digital software analysis of SDS-PAGE gels will yield somewhat different percentages of multimers compared to analytical SEC-HPLC of the same composition. Unless otherwise stated 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 in which 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, more 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 higher. 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 the fraction, unless otherwise specified. Thus, for example, band 2 contains a dimer of a homodimer, band 3 contains a trimer of a homodimer, etc.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 multimers" refers to multimers in 7 or more fractions, or fractions containing 7 or more mers of homodimers.

[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 tetramer fraction of homodimers constitutes 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, hi some embodiments, the tetramer fraction of homodimers constitutes 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 pentamer 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, hi some embodiments, the dimers of the pentamer 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 hexamer fraction of homodimers constitutes 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 hexamer fraction of homodimers constitutes 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 fraction comprises about 40% to about 60% of the total composition, the homodimer trimer to homodimer hexamer fraction comprises about 32% to about 50% of the total composition, the homodimer tetramer to homodimer hexamer fraction comprises about 30% to about 37% of the total composition, the homodimer pentamer to homodimer hexamer fraction comprises 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 in which approximately 80% of the total GL-2045 composition comprises homodimers or more (i.e., 2 or more bands). In some embodiments, approximately 60-80%, 62-80%, or 60-78% of the total recombinantly produced GL-2045 composition comprises homodimers or more (i.e., band 3 or more). In some embodiments, approximately 54-76%, 54-72%, 56-76%, or 54-67% of the total recombinantly produced GL-2045 composition comprises tetramers or more (i.e., band 4 or more). In some embodiments, a GL-2045 composition is provided in which 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 higher (i.e., band 6 or higher).

[0086] In some embodiments, recombinantly produced GL-2045 is provided in which 2-6 bands of the composition (i.e., homodimer dimer to homodimer hexamer) constitute about 39-61% or about 44-60% of the composition. In some embodiments, recombinantly produced GL-2045 is provided in which 3-6 bands of the composition (i.e., homodimer trimer to homodimer hexamer) constitute about 32-50% or about 35-48% of the composition. In some embodiments, recombinantly produced GL-2045 is provided in which 4-6 bands of the composition (i.e., homodimer tetramer to homodimer hexamer) constitute about 26-39% or about 30-39% of the composition. In some embodiments, recombinantly produced GL-2045 is provided in which 5-6 bands of the composition (i.e., homodimer pentamer to homodimer hexamer) constitute about 16-23% or about 18-23% 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 easily understand that the amount of homodimers or homodimers and dimers in the final product can be reduced using the optimized chromatographic methods described herein, or similar purification techniques. Thus, 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 the removal or reduction of a single multimer band, such as the 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 multimer 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 increase in percentage 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 fraction that was removed, depending on any variability due to the analytical method.

[0089] Pharmaceutical Compositions Administration of the GL-2045 compositions described herein will 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, intraarterial, subarachnoid, sublingual, oral mucosal, bronchial, lymphatic, intrauterine, subcutaneous, intratumoral, on an implantable device such as a suture, or integrated within an implantable device such as an implantable polymer, intradural, intracortical, or dermal. Such compositions will typically be administered as a pharma- ceutically acceptable composition as described herein. In a preferred embodiment, the isolated optimally manufactured 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. Any conventional media or agent is contemplated for use in the therapeutic composition, except insofar as it is incompatible with the vector or cell of the present invention. Supplementary active ingredients can also be incorporated into the composition.

[0091] The GL-2045 compositions of the present invention may be formulated in neutral or salt form. Pharmaceutically acceptable salts include acid addition salts (formed with free amino groups of proteins) formed with inorganic acids such as, for example, hydrochloric 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, for example, sodium, potassium, ammonium, calcium, or ferric hydroxide, and organic bases such as isopropylamine, trimethylamine, histidine, procaine, etc.

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

[0093] Further, one embodiment is a GL-2045 composition suitable for oral administration, provided in a pharma- ceutically acceptable carrier with or without an inert diluent. The carrier should be assimilable or edible, and includes 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 manufactured stradomer formulation contained therein, its use in an orally administrable optimally manufactured 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, and the like, 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 of skill in the art.

[0095] In certain embodiments, the GL-2045 composition in powder form is thoroughly combined or mixed with a semi-solid or solid carrier. Mixing can be performed in any conventional manner, such as by grinding. Stabilizers can also be added in 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, mannitol, protease inhibitors, and the like. More preferably, for orally administered compositions, the stabilizer can also include antagonists to gastric acid secretions.

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

[0097] In another embodiment, the GL-2045 composition in powder form creates nanoparticles that encapsulate the immunologically active biomimetic or is thoroughly combined or mixed with the material 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 the 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 particular GL-2045 formulation that may be used is a solution of immunologically active biomimetic protein in a hypotonic phosphate-based buffer containing 6 mM sodium phosphate monobasic monohydrate, 9 mM sodium phosphate dibasic heptahydrate, 50 mM sodium chloride, potassium-free at pH 7.0+ / -0.1. The concentration of the immunologically active biomimetic protein in the hypotonic buffer may range from 10 μg / mL to 100 mg / mL. This formulation may 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 the formation of 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, the 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 / volume for application to the skin for the treatment of diseases on or beneath the skin. As used herein, the term "topical administration" includes application to dermal, epidermal, subcutaneous, or mucosal surfaces.

[0101] In addition, GL-2045 compositions can be formulated into polymers for subcutaneous or subcutaneous implantation.Preferred formulations for implantable drug-injection 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). One of ordinary skill in the art will know that many similar polymers and hydrogels can be formed that incorporate stradomers affixed 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 bring about the improvement or repair of symptoms.The formulation is easily administered in various 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 in any case determine the appropriate dose for each individual subject.In addition, for administration to humans, the formulation meets 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 disease 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 particular 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 longer intervals. A biphasic dosing regimen may be used, with a 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 administration of one or more additional medical and / or therapeutic agents. In further embodiments, the additional pharma- ceutically active agent comprises a steroid; a biologic anti-autoimmune drug, such as a monoclonal antibody, a fusion protein, or an anti-cytokine; a non-biologic anti-autoimmune drug; an immunosuppressant; an antibacterial; and an antiviral drug; a cytokine; or an agent that can otherwise act as an immunomodulator. In yet 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, Pertuzumab, brentuximab, ipilumumab, denosumab, canakinumab, ustekinumab, catumaxomab, ranibizumab, panitumumab, natalizumab, bevacizumab, cetuximab, efalizumab, omalizumab, toitumomab-I131, alemtuzumab, gemtuzumab, trastuzumab, palivizumab, basiliximab, daclizumab, abciximab, murononomab, vedotin, ibritumomab tiuxetan, motavizumab, or certolizumab. 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 yet further embodiments, the immunosuppressant is cyclosporine A, tacrolimus, sirolimus, mycophenolate mofetil, everolimus, OKT3, antithymocyte globulin, basiliximab, daclizumab, or alemtuzumab. In yet further embodiments, the optimally manufactured stradomer is administered before, during, or after administration of a chemotherapeutic agent. In yet 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 after administration with the additional therapeutic agent.

[0106] In one embodiment, the GL-2045 composition is administered covalently attached to an implantable device. In one embodiment, the optimally manufactured stradomers are attached to sutures. In another embodiment, the optimally manufactured stradomers are attached to grafts or stents. In another embodiment, the optimally manufactured stradomers are attached to heart valves, orthopedic joint replacements, or implanted electronic leads. In another embodiment, the optimally manufactured stradomers are attached to and embedded within an implantable matrix. In a preferred embodiment, the optimally manufactured stradomers are attached 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 stradomers are administered attached within a hydrogel with a pore size large enough to allow entry of immune cells to interact with the attached stradomers 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 with 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 via 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 polymyositis, Neuropathy, gangrenous fasciitis, pemphigus, gangrene, dermatomyositis, granuloma, lymphoma, pneumonia, 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, especially associated with type 1 rhus, asthma, epidermal necrolysis, Lambert-Eaton myasthenic syndrome, neuropathy, uveitis, Guillain-Barre syndrome, graft versus host disease, stiff man syndrome, paraneoplastic cerebellar degeneration with anti-Yo, paraneoplastic encephalomyelopathy and sensory neuropathy with anti-Hu, 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, muscular dystrophies. As used herein, the term "intravenous administration" includes all techniques for delivery to the systemic circulation via intravenous infusion or drip.

[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 Produced 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 function as an important biopharmaceutical for treating inflammatory diseases and disorders, and for modifying immune function in various other contexts, 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 medical conditions currently being treated 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 include those currently routinely treated with broad spectrum immunosuppressive therapy, such as autoimmune cytopenias, chronic inflammatory demyelinating polyneuropathy, Guillain-Barré syndrome, myasthenia gravis, anti-factor VIII autoimmune disease, dermatomyositis, vasculitis, and uveitis, in which hIVIG has been found to be 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 may be used or are already in clinical use. Conditions that may be effectively treated by the compounds of interest in this invention include inflammatory diseases with an imbalance in the cytokine network, autoimmune diseases mediated by pathogenic autoantibodies or autoaggressive T cells, or chronic relapsing autoimmune, inflammatory, or infectious diseases or processes in the acute or chronic phase.

[0121] In addition, other medical conditions having an inflammatory component involving complement would benefit from treatment with GL-2045 compositions such as those believed to be associated with 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 believed to be associated with post-viral encephalitis including Rasmussen's syndrome, West syndrome, and Lennox-Gastaut syndrome.

[0122] A 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 may 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 manufactured 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 sense of well-being. Thus, one of skill in the art will understand that a treatment may improve a disease condition, but may not be a complete cure for the disease. Specifically, improvement in a subject may include reduced autoimmunity as evidenced by one or more of: reduced inflammation; reduced inflammatory laboratory markers such as C-reactive protein; improved autoimmune markers such as autoantibodies or improved platelet, white, 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 frequency and volume of muscle cramps and diarrhea; 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 the improvement or repair of 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 is more than one "therapeutically effective dose" of GL-2045.

[0125] As used herein, "prophylaxis" can mean preventing the symptoms of a disease altogether, delaying the onset of symptoms of a disease, or reducing the severity of symptoms of a disease that subsequently develops.

[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). The 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 in which the optimally engineered stradomers of the present invention may be used include anti-glomerular basement membrane antibody-mediated nephritis, including Good-Bascher'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-body syndrome or hyperkinetic startle syndrome; antiphospholipid, anticardiolipin, and anti-β2-glycoprotein I antibodies in recurrent spontaneous abortion, Hughes syndrome, and systemic lupus erythematosus; anti-glutamic acid decarboxylation in stiff-body syndrome, autoimmune cerebellar ataxia, or limbic encephalitis. acid enzyme antibodies; anti-NMDA receptor antibodies in a newly described syndrome that includes both limbic and subcortical features with prominent movement disorders common in young adults and children that are often associated with ovarian teratomas but 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; 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 matrix; 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, renal cancer, and rectal cancer; bone metastases, bone pain management, and humoral malignant hypercalcemia, ankylosing spondylitis and other spondyloarthropathy; transplant rejection, viral infections, hematological 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, etc.) Neoplasms of lymphoid precursor cells, including acute lymphoblastic leukemia / lymphoma, B-cell acute lymphoblastic leukemia / lymphoma, and T-cell acute lymphoblastic leukemia / lymphoma; neoplasms of mature T and NK cells, including thymoma, peripheral T-cell leukemia, adult T-cell leukemia / T-cell lymphoma, and large granular lymphocytic leukemia; neoplasms of myeloid cells, including 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 histological disorders, 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 vasculature (angiosarcoma and hemangiopericytoma), or other cancers.

[0129] The GL-2045 composition of the present invention can be used to treat autoimmune diseases. As used herein, the term "autoimmune disease" refers to a diverse group of more than 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, endocrine system, skin, blood, and 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 invention may be a hematological immunological 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, erythroblast aplasia, Diamond-Blackfan anemia, hemolytic disease of the newborn, immune-mediated neutropenia, refractory 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-Barre syndrome, chronic inflammatory demyelinating polyneuropathy, 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 autonomic neuropathy.

[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, at the same time, 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 abortions, 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 skin blistering 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 vacuolar 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, cryptopathic cirrhosis, reactive arthritis, Crohn's disease, Whipple's disease, ulcerative colitis, or sclerosing cholangitis.

[0137] The disease or condition may also be graft versus host disease, antibody mediated graft rejection, post bone marrow transplant rejection, infectious post 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 responses 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 bite allergies. Exemplary allergies include urushiol in poison ivy and oak; house dust antigens; birch pollen components Bet v 1 and Bet v 2; 15 kD antigen in celery; apple antigen Mal d 1; Pru p3 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 Chinese dwarf grass; house dust mite Der p 1, Der p2, or Der f 1 for house dust mite allergy; α-gliadin and γ-gliadin epitopes in gluten; bee venom phospholipase A2; 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 the optimally manufactured stradomer(s) for a period of time, 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 the 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 easily applied to modify immune system responses in a variety of contexts to affect specific changes in immune response profiles. Modification or regulation of immune responses in a subject refers to increasing, decreasing, or changing the proportion or components of immune responses. 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 complement and interact with these receptors. Antibody production can also be increased or increased; the proportion of two or more cytokines or immune cell receptors can be changed; or additional types of cytokines or antibodies can be produced.

[0141] In a preferred embodiment, subjects with an autoimmune or inflammatory disease have their immune response altered, comprising administering to the subject a therapeutically effective amount of a GL-2045 composition as described herein, the therapeutically effective amount of the GL-2045 composition altering the immune response in the subject. Ideally, this invention treats a disease or condition in a 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 CCL, CXC, XC, and FAM19 chemokine family members. In a preferred embodiment, IL-6 or IL-8 is reduced in response to treatment. In a particularly preferred embodiment, 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 biomimetic.The altered immune response can be an altered autoantibody level in the subject.The altered immune response can be an altered autoaggressive T cell level in the subject.

[0142] For example, reduced amounts of TNF-alpha production in autoimmune diseases may have therapeutic effects. A practical application of this is anti-TNF-alpha antibody therapy (e.g., REMICADE®), which has been clinically proven to treat psoriasis vulgaris, rheumatoid arthritis, psoriatic arthritis, Crohn's disease, ulcerative colitis, and ankylosing spondylitis. These autoimmune diseases have different etiologies, but share a key 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 could also be direct or indirect modulation to affect antibody production, e.g., autoantibodies that target the subject's own tissues, or altered auto-aggressive T-cell levels in the subject. For example, multiple sclerosis is an autoimmune disorder involving autoreactive T cells that can be treated by interferon beta therapy. See, e.g., Zafranskaya M, et al., Immunology 2007 May;121(l):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 may be used to modulate the expression of costimulatory molecules from immune cells, including dendritic cells, macrophages, osteoclasts, monocytes, or NK cells, or to inhibit 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 may 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 immune cells are exposed to the optimized immunologically active biomimetic in vitro, further comprising a step of 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 amelioration of the autoimmune disease.

[0144] The GL-2045 compositions described herein may also be used as components of devices. In some embodiments, the GL-2045 provided herein may be coated on devices such as medical implants. For example, an optimally manufactured stradomer may be coated on a coronary stent or as part of a nanoparticle therapy to improve penetration and prolong drug release, for example, for intraocular use in uveitis or macular degeneration. The optimally manufactured stradomers described herein may also be used as a component of a diagnostic. In some embodiments, one skilled in the art may personalize treatment by determining in which patients the use of a stradomer may be particularly beneficial. For example, one skilled in the art may 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. EXAMPLES

[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, aspects of the following upstream manufacturing processes 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 in which the optimal multimerization profile of GL-2045 was maintained. The following examples are provided by way of illustration only and not by way of limitation.

[0147] Example 1 - Fractionation and Biolayer Interferometry Analysis of GL-2045 A solution of GL-2045 was fractionated using a GE Hi-Load 26 / 60 Superdex 200pg column (GE, #17-1071-01) in 0.05M Tris-HCL + 0.15M NaCl buffer (pH 7.5). 3.2mL of GL-2045 solution was loaded at a flow rate of 2.6mL / min. Six fractions (1-6) were collected in a volume of 2.0mL and protein concentration was determined by UV measurement at 280nm (Figure 1A). Multimerization for each of the GL-2045 fractions was evaluated. Briefly, samples of each of fractions 1-6 were loaded onto 4-12% non-reducing Nu-Page BT gels (Invitrogen, #NP0322BOX). The samples were run at 150 volts for approximately 3 hours. The results are provided in FIG. 1B and demonstrate a clear difference in the presence of higher multimers of GL-2045 in fractions 1-6. Fractions 1-3 are composed of lower 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% dimers of 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. When binding occurs, it produces an increase in optical thickness at the biosensor chip, which results in a wavelength shift (detected as a response unit of "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 assay buffer from ForteBio (Cat.#18-1092) for 300 seconds. The loaded sensor was transferred into 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 into 1× kinetic buffer containing purified stradomers of choice 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 into 1× kinetic buffer for 300 seconds, and RU values, on-ratio values, dissociation ratios, and Kd values ​​were calculated using ForteBio software.

[0150] The binding curve results are shown in Figure 2, and the 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 the 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 was analyzed for multimerization on non-reducing gels (Figure 3B). To determine the effect of each fraction on complement activation, CD20-expressing Wil2 cells were cultured with 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 in medium 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 for 15 minutes at room temperature. 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) show more pronounced inhibition of CDC than the lower molecular weight multimers present in fractions 1-4. It is also noted that only fractions containing 4 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. The supernatant of GL-2045 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). The affinity chromatography-purified GL-2045 was stored in 1X 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 polished by cation exchange chromatography on a POROS CIEX column (1.2 cmD x 10 cmL GOPURE column, Poros XS, Life Technologies, #4448885) with a binding buffer of 50 mM sodium acetate, pH 5.0, and an elution gradient (0-100% elution buffer) with 50 mM sodium acetate, pH 5.0, 1 M NaCl. This polishing step was performed without removing the highest order multimers and / or disordered aggregates from the final fraction. As a final step, the CIEX polished GL-2045 was concentrated to a volume of ≦5 mL, buffer exchanged against gel filtration running buffer, and injected into 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 (Figure 5B and Figure 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 (Figure 6A and Figure 6B). The EC50 values ​​for each fraction are shown in Table 3. These results demonstrate that the higher order multimers (fractions 1C4, 1C5, 1C6, 1C7, 1C8, 1C9, 1C10, and 1C11) demonstrate more avid binding, noted by a lower EC50, to FcγRIIIa than the lower order multimers (fractions 1D9, 1E4, and 1F7), and surprisingly, more avid binding than the highest molecular weight multimers (fractions 1C3, 1C2, 1C1, 1B12, 1B11). Presuming that the very highest molecular weight fractions contain 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, demonstrate that all of the GL-2045 These data show that the high molecular weight fraction of 1B11 and 1B12 do not demonstrate increased binding to FcγR, likely due to the effect of disordered assembly of homodimers as opposed to the formation of highly ordered, high molecular weight multimers. These data indicate the need for optimized downstream production methods (including optimized conditions for Protein A purification, ion exchange chromatography, and hydrophobic interaction chromatography) in combination with optimized upstream production methods to result in the generation and retention of higher molecular weight, higher order multimers, and the removal of disordered, high molecular weight aggregates (e.g., 1B11 and 1B12), which are less effective at binding to the low affinity receptors of their targets (see EC50 values ​​for 1B11 and 1B12 in Table 3). [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 at pH 7.2. After a first wash with binding buffer and a second wash with a buffer containing 1 M NaCl, 5 mM EDTA, 2 M urea, 50 mM phosphate at pH 7.0, the protein bound to pA was eluted with 0.1 M glycine at pH 2.7.

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

[0157] These purified fractions were utilized 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. Prior to addition to the Boyden chamber, neutrophils (purified from whole blood from PBMCs, final concentration 2.25 x 106 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 to determine the percentage chemotaxis for each condition (Figure 8). No chemotaxis was observed for GL-GLM-001 (recombinant Fc control, G001). Higher order multimers (containing 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 (containing 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 the production of GL-2045 The objective of this experiment was to test the effect of a panel of basal media on GL-2045 protein titer, cell viability, cell density, and multimerization of GL-2045.

[0160] GL-2045 was grown in ProCHO5 medium (Lonza #12-766Q) containing L-glutamine (Lonza #17-605E) and sodium hypoxanthine and thymidine (HT, Gibco #11067-030) in a shaker incubator at 37°C and 5% CO2. After passage, cells were washed and inoculated in duplicate at 0.5 x 106 cells / mL in the selected medium (shown in Table 4) in 50 mL tube spins. 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 rotation speed. 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 L-glutamine and HT as ingredients, as well as 4 mM L-glutamine and 1× sodium hypoxanthine and thymidine. Growth conditions for selected media are shown in Table 4. [Table 4]

[0161] GL-2045 cell cultures grown in selected media were assessed for cell density, cell viability, protein titer, and percentage of higher order multimers. Cell density and cell viability assessments were performed 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 for all media except ActiCHO P, BalanCD CHO, ExpiCHO, Cell Vento CHO 210, and Cell Vento CHO 210 compared to day 4. Of the 19 media, the greatest cell viability was observed on day 8 with ActiCHO P, followed by ExpiCHO, Cell Vento CHO 110, and HYQ SFX-CHO LM tested. Of the 19 media, the only medium with a positive trend in cell viability from days 4 to 8 was ActiCHO P. Thus, the only medium that produced high cell density on day 4 and did not have a negative trend for cell density on day 8 was ActiCHO P. [Table 5] [Table 6]

[0162] Cultures were pelleted on day 10, filtered at 0.2 μm, 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) with 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 1× PBS, pH 7.0 through a HiPrep26 / 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 titers were observed at day 10 with Cell Vento CHO 110, followed by BalanCD CHO Growth A medium, ActiCHO P, and ExpiCHO. A significant drop in titer occurred with the other media. [Table 7]

[0163] To determine the percentage 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. Samples were heated at 80°C for 10 min, loaded onto the gel, and run for 1 h 25 min at 150 V using running buffer (Tris Acetate SDS (20X) (Life Technologies, #LA0041)). Gels were washed in DI water, stained with SimplyBlue Safe (Life Technologies, #LC6060), and destained with DI water. After complete destaining, images were cropped using a G:BOX system from Syngene and the banding patterns were analyzed by densitometry using the GeneTool of Syngene software. The intensity of each individual band in each lane was measured (Figure 12A and Figure 12B).

[0164] Unexpectedly, we observed the highest percentage of higher order multimers above band 4 of 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 must 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 - Media screening of GL-2045 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 titer and the three basal media associated with the lowest GL-2045 protein titer were subjected to a repeat experiment in which commercial feed was provided during cultivation. Cell Vento 110 and ExpiCHO, which produced high titers, were not selected for feeding experiments because the manufacturer's recommended feed was not identified. Cell Vento CHO 110 is a complete medium that should be used for cell adaptation without feeding, and Cell Vento 210 is used for cultivation 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 sodium hypoxanthine and thymidine (HT, Gibco #11067-030) in a shaker incubator at 37C and 5% CO2. After passaging, cells were washed and inoculated at 0.5x106 cells / ml, then subcultured when the density reached 1x106-3x106 cells / mL and ≥80% viability. GL-2045 clone 58 was directly adapted 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) were obtained for at least 2-3 passages. Cells were seeded at 0.5×106 cells / mL in selected media (d0) 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 for all cultures except PowerCHO3 CD (d3). Feeding for PowerCHO3 CD medium began on day 1. 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 manufacturer recommended feeds according to the recommended protocol. Feeding strategies and schedules for each of the tested media are outlined in Figure 13A and Figure 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 measurements of protein in cell supernatants were performed by biolayer interferometry (Octet) of cell supernatants 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-fold compared to all other media / feed combinations tested, except for Cell Vento 210 (FIG. 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 in other media tested with the manufacturer's recommended feeding, 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 diet A and ActiCHO diet B developed substantially higher titers than any other medium and manufacturer's recommended feeding tested (FIG. 16). Titers generated from ActiCHO P cultures were at least 4-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 percentage multimers in bands 5 or higher (5+) by 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 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 in terms of cell density, viability, and protein titer. Densitometry analysis shows that CD FortiCHO medium + CD Efficient Feed C has the highest percentage of maximum 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 even higher order multimer bands above band 4 at 30.1% with negligible amounts of nonspecifically aggregated high molecular weight material that does not migrate in the gel. Therefore, ActiCHO P is likely to have the highest percentage of fully functional higher order multimer bands. These data further indicate that upstream manufacturing conditions not only affect cell viability, density, and total protein titer, but also affect the generation of clinically effective higher order multimers of GL-2045. As demonstrated in Example 3, the highest molecular weight fractions exhibit reduced binding to FcRγIIIa, suggesting that the highest molecular weight fractions (e.g., disordered GL-2045 aggregates) are less biologically active than highly ordered multimers of GL-2045. As shown herein, it is very surprising that, 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 produced optimal protein titers and production of higher multimers, modified feeding schedules were tested to determine whether similar or optimized results could be obtained by feeding every other day. As demonstrated in FIG. 18, feeding every other day (blue) did not significantly affect cell density (FIG. 18A), cell viability (FIG. 18B), culture pH (FIG. 18C), or protein titer (FIG. 18D) compared to daily feeding. These results surprisingly indicate that feeding every other day can produce similar results and may be preferable to maintain sterility and minimize production costs. However, similar experiments with feeding every third day suggested that viability and protein production may begin to decrease with less frequent feeding than every other day (FIG. 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 inoculated at 0.3 x 106 cells / ml in ActiCHO P complete medium ActiCHO P + 6 mM L-glutamine (Lonza, #17-605E) or Power CHO-2 CD complete medium and cultured without temperature shift. ActiCHO P PowerCHO cultures with +1 mL of ActiCHO feed A (PAA, #U15-072) + 0.1 mL of feed B (PAA, #U05-034) were fed daily. 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 PowerCHO2 and ActiCHO P generate the same cell viability and GL-2045 protein yield when used with ActiCHO feeds A and B according to the manufacturer's recommendations. Thus, ActiCHO feeds A and B may 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 production of GL-2045 The purpose of this experiment was to evaluate the optimal timing and extension of the temperature shift. Numerous investigators have examined the effect of temperature shift on 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 investigators have surprisingly found that a minimum viable cell density of 10 million cells / mL is required at the time of the temperature shift for optimal GL-2045 expression and maximum titer. Moreover, this requirement is more critical than the day of culture at which the temperature shift occurs. The timing of the temperature shift is surprisingly most successful when the viable cell density is in the logarithmic growth phase, generally between 10 and 15 million cells / mL. This generally corresponds to day 4-5 of the bioreactor culture, depending on the initial seeding density.

[0177] Further departing from what is described in the art, for optimal upstream conditions to produce high titer GL-2045, a temperature shift of 37 degrees Celsius (37°C) + / - 1.0C to 32.5C + / - 1.0C is preferred over a temperature shift to 31°C + / - 0.5C. 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 10L 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 280ml of PAA Feed A (cat#U15-053) daily and 28ml 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 steps GL-2045 was purified by affinity chromatography (AC) with a Protein A HiTrap MabSelect SuRe column (GE#11-0034-95) with 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. 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 diluted sodium hydroxide (0.1-0.5 M NaOH) through the column between purification cycles to hydrolyze precipitates while sanitizing the Protein A resin, thus 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 homodimer dimers, after the second purification run (Figure 22). As shown in Table 12, densitometry analysis of SDS-PAGE demonstrated 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 the lower molecular weight band indicates that there is an avidity-based binding to the affinity column, where the high molecular weight multimeric GL-2045 containing multiple Protein A binding sites outweighs the lower molecular weight species, causing the loss of the lower molecular weight species and effectively altering the composition of the drug. These data suggest that more frequent CIP procedures, and therefore 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 deteriorate the Protein A columns most commonly used in the art. Thus, more frequent use of such buffers would result in more rapid deterioration of the Protein A column. Thus, a Protein A column that can withstand frequent CIP procedures with high-strength NaOH buffers, such as 0.5M NaOH, must be used for regeneration of the 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, thus 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 regular daily purification using a HiTrap MabSelect column (GE #28-4082-58) resulted in a noticeable change in the composition of purified GL-2045 after approximately 6-7 purification runs, with a slight loss of homodimer and dimer fractions. For example, one skilled in the art of 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 solve this problem, the manufacturer's recommended CIP procedure with 0.1 M NaOH was performed 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 used in the art. Although the use of frequent CIP procedures provided some improvement, the problem of loss of lower molecular weight species was not resolved. We therefore reasoned that the avid binding of GL-2045 to Protein A required a more stringent CIP regimen than one skilled in the art would typically use for complete regeneration of the column to promote retention of homodimers and dimers.

[0182] However, the resins commonly used in Protein A columns (e.g., MabSelect) are not NaOH-resistant and therefore deteriorate quickly with the use of more stringent NaOH buffers, and such regeneration is associated with reduced purification capacity and changes in GL-2045 multimer composition.However, less commonly used Protein A media (e.g., MabSelect SuRe (General Electric #11-0034-95)) can withstand improved cleaning with 0.5 NaOH.Thus, we used MabSelect SuRe columns with daily CIP procedures using 0.5 M NaOH buffer.After performing more frequent and more stringent CIP procedures, 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 of skill in the art working with monoclonal antibodies or Fc fusion proteins in order to retain 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 A pH elution gradient is commonly used with Protein A columns during protein purification to optimize for total protein yield, but is typically not used to alter the composition of the drug. We have discovered that such a pH elution gradient on a Protein A column can be used to remove disordered aggregates of GL-2045 from higher order multimers. The supernatant of GL-2045 CHO was purified by affinity chromatography (AC) with Protein A HiTrap MabSelect SuRe (GE#11-0034-95) containing a mixed buffer of 20 mM sodium phosphate, 0.15 M NaCl at pH 7.2, followed by an additional wash with binding buffer. GL-2045 bound to Protein A was eluted by an elution buffer containing 0% to 100% 0.1 M glycine at 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 into each well. The equivalent protein amount of each of the fractions was then run on a non-reducing SDS-PAGE gel (4-12% NuPage Bis-Tris, Invitrogen #NPO322BOX).

[0185] SDS-PAGE analysis of fractions obtained by elution of Protein A column with 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 main species of GL-2045 (e.g., homodimers, dimers, and higher multimers) by pH gradient elution of Protein A affinity column. As shown in Examples 1-3, these fractions may represent less active species, as demonstrated 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 routinely used for the polishing step of monoclonal antibody and Fc fusion protein purification. We tested a cation exchange medium, POROS CIEX (Invitrogen GoPure XS (10 mL) cat#4448885), with 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 washing buffer. The effect of elution buffer (EB) salt content on the polishing of GL-2045 was tested using a 50 mM sodium acetate elution buffer with variable amounts of added buffer B (1 M NaCl at pH 5 shown as % in Figure 24). Chromatography runs were performed on an Akta Avant. Briefly, the Avant method involves equilibrating a CIEX column with 50 mM sodium acetate at 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 at pH 5, was loaded onto the column. 5 cv of binding buffer was used to wash the column 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 (Figure 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 separation of GL-2045 molecular weight species when eluted with 30% EB, 40% EB, or 50% EB elution buffers. 91.7% of the material was eluted with elution buffer containing 30-40% buffer B, and the only material recovered with elution buffer containing 50% buffer B was high molecular weight material. These data demonstrate that changing the elution buffer can substantially denature the multimeric composition of GL-2045. Thus, it is clear that the elution buffer selected for ion exchange can be used to denature the multimeric composition of GL-2045, which is a novel use of this technology. At the same time, the inventors have discovered that if changes in the composition of GL-2045 are not desired in the ion exchange polishing step, a level of precision not typically practiced by those skilled in the art is required in the selection of the salt concentration of the elution buffer for use in the ion exchange polishing step.

[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 elution buffer containing 30-40% buffer b, and the only material recovered with elution buffer containing 50% buffer B was high molecular weight material, elution buffers with a range of 30-40% buffer b were selected for further analysis. The method used was similar to that described in Example 11.

[0192] SDS-PAGE analysis demonstrated a dramatic difference in the separation of GL-2045 homodimers when eluted with elution buffers containing 35%, 36%, or 37% or more of buffer b (35% EB, 36% EB, 37% EB, etc., respectively). Note that the homodimers and homodimer dimers clearly visible at 35% EB are, in contrast, greatly reduced at 36% EB and completely removed at 37% EB or more (Figure 25). Similarly, SDS-PAGE analysis demonstrated a dramatic difference in the separation 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. It is therefore evident that the elution buffers selected 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 step elution can be used by one skilled in the art to select a single step or multiple step elution to obtain a desired GL-2045 profile. For example, polishing GL-2045 using a POROS CIEX column and an elution buffer containing 36.5-38.5% buffer b will retain all homodimers, dimers, and multimers through multimer 10, and will elute the 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 Producing an optimal composition of GL-2045 requires removing material above 1000 kD to minimize the amount of the highest order GL-2045 multimers (e.g., bands above clearly delineable band 10, approximately 600 kD) and to remove both large homodimeric aggregates and the highest order multimers, where increasing valency poses an increased theoretical 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 % 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 remaining high molecular weight material.

[0195] The GL-2045 peaks were quantified by densitometry (summarized in Table 15 as percentage intensities in SDS-PAGE bands, FIG. 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 with acetate elution buffers containing 38% or 39% buffer B at pH 5 yielded approximately 85% of the preferred fraction of GL-2045 and reduced the higher molecular weight fractions above 600 kD. Visual inspection of the SDS-PAGE showed that high molecular weight material was lowest in fractions eluted with elution buffer containing 38% buffer b, while densitometric analysis showed 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 in the high molecular weight fraction (band 1) compared to material purified by affinity chromatography alone. Overall, the elution analysis suggests that the amount of large aggregates and highest order multimers can be controlled by applying controlled elution conditions with the POROS CIEX column. It is therefore evident that the elution buffer selected for ion exchange can be used to denature large aggregates and the most highly ordered multimeric compositions of GL-2045, a novel use of this technique. 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 a week and filtered at 0.2 μm into a 1 L filter system (Corning #431098). GL-2045 supernatants were then purified by Protein A HiTrap MabSelect SuRe (GE#11-0034-95) with a binding buffer of 20 mM sodium phosphate, 0.15 M NaCl at pH 7.2, and eluted with 0.1 M sodium citrate elution buffer with a pH of 3.0-3.6. AC-purified GL-2045 was stored in 1X PBS at pH 7.0 (Quality Biological, Inc. #119-069-101).

[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 the 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 (start buffer) and 3.5 mg of AC purified GL-2045 (diluted in 4 volumes of start buffer) was loaded onto each column. After washing with start 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 into 15% Tris HCl (Bio-Rad #161-115). Staining was performed using a silver stain kit form Invitrogen #LC6100.

[0200] The seven different HIC columns demonstrated different multimer profiles of GL-2045. As an example, the Butyl HP column separated the homodimer fraction (fraction A10) from the multimeric 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 during the elution fractions with the Octyl FF column, indicating that it may be well adapted to a flow-through mode polishing method for GL-2045. Furthermore, the fractions eluted from the Octyl FF column contained higher molecular weight species, indicating that the column may also be well adapted to the removal of very high molecular weight homodimeric aggregate species with lower efficacy.

[0201] A similar experiment was performed on the mouse version of GL-2045, known as M045, where 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, 1 M ammonium sulfate at pH 7.0 (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 elution buffer at pH 7.0 by gradient elution (Figures 30-31).

[0202] HIC purified fractions of M045 were analyzed by SDS-PAGE to determine the effect of HIC column polishing on the M045 multimer profile. These results further demonstrated that a hydrophobic interaction column can be used to denature the multimer 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 of the upstream and downstream manufacturing process of GL-2045 that result in optimization of (1) protein titer, (2) cell viability throughout culture, and (3) multimerization of GL-2045, and (4) maintenance of the multimer profile in the final GL-2045 drug substance. Importantly, the level of multimerization of GL-2045 is essential for the clinical efficacy of the stradomer (see Examples 1-4). Current culture methods do not necessarily aim at optimized production of specific fractions or enhancement of specific multimerization patterns. Thus, the upstream culture reagents and conditions as well as downstream purification media and conditions that affect multimerization are all unknown 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 base medium was ActiCHO P. CHO cells cultured in a bioreactor in ActiCHO P base medium resulted in high cell density and cell viability while being optimized for increased protein titer compared to other base media tested. 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 feed for generating optimally produced GL-2045 is ActiCHO P and ActiCHO Feed A and Feed B. The data described herein further demonstrate that the optimal feed was ActiCHO P Feed A and Feed B, which were added to the culture daily or every other day. ActiCHO P Feed A and Feed B maintained high cell density and high cell viability while resulting in a protein titer that was 4-fold greater than other medium / feed combinations tested. Importantly and unexpectedly, ActiCHO P medium with Feed A and Feed B resulted in high levels of highly ordered multimers and, importantly, a reduced percentage of high molecular weight disordered aggregates of GL-2045 compared to 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 mitigate the risk of contamination associated with daily culture operations.

[0207] Furthermore, ActiCHO P medium with diets A and 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 to generate optimally produced GL-2045 is a 37°C to 32.5°C shift based on cell density. The data described herein additionally demonstrates that a 37°C to 32.5°C temperature shift 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 simply describe a 37°C to 31°C temperature shift based on culture days. We unexpectedly found that a shift in temperature to 32.5 °C after cells reached a density of approximately 10-15 x 106 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 show that certain downstream purification protocols result in GL-2045 compositions with optimized multimerization profiles. In carrying out these purification methods, strict care must be taken to maintain the desired multimer 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 the primary goals.

[0210] (4) Optimized protein A purification of GL-2045 requires frequent and stringent CIP procedures. GL-2045 binds avidly to protein A. This avid binding resulted in GL-2045 remaining bound to protein A media in the column when the CIP procedures commonly used for mAb purification were used. As a result, repeated cycles of using protein A columns resulted in the homodimeric fraction of GL-2045 being 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 0.5M 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 some Protein A columns, specifically MabSelect SuRe, can be used for purification of GL-2045 and will require frequent CIP procedures with approximately 0.5M 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 multimers. The use of a pH elution gradient with purification of Protein A resulted in the highest molecular weight components eluted in the first and last elution fractions. These data indicate 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 disordered, high molecular weight aggregates that were previously shown to have reduced biological activity.

[0212] (6) The optimal elution buffer for polishing GL-2045 by ion exchange chromatography is acetate buffer + 30-40% buffer b, specifically 37.5%-39% + / - 0.5%. Ion exchange chromatography is commonly used to polish drug impurities during mAb production. However, the inventors used ion exchange chromatography to remove specific fractions of GL-2045 (e.g., the highest order multimers of homodimers and high molecular weight disordered aggregates) such that an optimal multimerization profile was achieved. Specifically, the inventors 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 specifically maintained the amount of homodimers present in the final GL-2045 product while also optimizing for reduced amounts of disordered aggregates.

[0213] (7) A hydrophobic interaction column (HIC) can be used to achieve a specific GL-2045 multimerization profile. The data herein demonstrates that multiple HICs can be used in the polishing step of purifying GL-2045. 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 a more desirable outcome. Alternatively, a HIC column can be used in a binding 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 the 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. PiggyBac transposons contain inverted terminal repeats that have preferential insertion into highly transcribed regions of the genome and, in addition, provide insulation by gene suppression. Transfection results in integration of the expression cassette into highly transcribed genomic regions, thereby building a bank of stably transfected CHO cells with fewer than 20 genomic insertions of the transgene. The stably transfected CHO cells were then cultured in a bioreactor with 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 >95% on day 18 and >80% on day 21 of culture, as well as final total protein titers of >9,000 mg / mL, with >70% of the GL-2045 present as non-homodimers and >30% as higher order multimers above the fifth multimer.

[0217] GL-2045 was harvested from the culture supernatant by a tangential flow filtration system that does not interfere with the passage of the most highly ordered multimers, thereby preserving the homodimer and multimer profile of the supernatant. Downstream manufacturing methods were then used to isolate GL-2045, remove impurities, and isolate specific fractions to control the multimer profile of GL-2045 (e.g., removal of 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 high alkaline regeneration. Furthermore, more than one washing buffer was used to allow further control over the purification process. In addition, CIP procedures were 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 were performed with 0.5M NaOH buffer to remove GL-2045 multimers that were avidly bound to the column. GL-2045 was eluted from the Protein A column with or without a pH elution gradient. After purification by affinity chromatography on the Protein A column, an additional polishing step 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 a HIC column. Octyl FF resin was used as an additional polishing step to remove high molecular weight disordered aggregates of GL-2045. 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 polishing step, especially in flow-through mode.

[0218] While 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 produced GL-2045 made by the methods described herein, GL-2045 was produced in a bioreactor according to the upstream methods described herein using ActiPro basal medium, feeding, temperature shifting as described herein. The resulting GL-2045 supernatant was then sent 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 evaluated 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 blue dots in Figure 33) and then purified with 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 reach 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 step of analytical HPLC, the percentage of homodimer, dimer, trimer, tetramer, pentamer, hexamer, and 7+mer fractions was evaluated. Briefly, the supernatant of GL-2045 stably transfected CHO was generated according to the upstream method described herein. Then, GL-2045 was 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. Samples were compared by analytical SEC-HPLC. Briefly, isocratic separation was performed by HPLC using a series of two SEC columns (Agilent Bio SEC (300 Å)) with UV detection at 280 nm on a high performance liquid chromatography system (Agilent 1100 HPLC system). Chromatography is performed with a run time of 60 min and a flow rate of 0.5 mL / min. The relative area percentages of each peak are calculated.

[0221] The results are shown in Figure 33. As is evident in Figure 33, downstream processing of GL-2045 altered the smallest fractions, homodimer and homodimer dimer, and the largest fraction, 7mer+, with fractions 3-6 remaining 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 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 that comprised 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 method for producing a composition comprising a homodimer and a multimer of said homodimer, said homodimer comprising two monomers each comprising amino acids 21-264 of SEQ ID NO:4, said multimer of said homodimer comprising at least about 80% of the total composition; The method (a) culturing Chinese hamster ovary (CHO) cells stably transfected with an expression vector comprising a nucleic acid sequence encoding SEQ ID NO:4 in a medium at a growth temperature of 37° C.±1° C. until the cells reach a density of about 5 million to about 30 million cells / mL; (b) shifting the growth temperature from 37° C.±1° C. to 32.5° C.±1° C.; (c) harvesting said homodimers and multimers of said homodimers from said culture medium.

2. The method described in claim 1, further comprising shifting the pH of the medium from 7.1 to 7.0 on the fifth day of culture.

3. 3. The method of claim 1 or 2, wherein the cells are grown to a density of about 10 to about 25 million cells / mL, about 10 to about 15 million cells / mL, or about 15 to about 20 million cells / mL prior to shifting the growth temperature.

4. 4. The method according to any one of claims 1 to 3, wherein the culturing is carried out in ActiCHO P or in ActiCHO P basal medium containing glutamine, and the CHO cells are fed ActiCHO feed A and ActiCHO feed B, or HyClone Cell Boost 7a and HyClone Cell Boost 7b during the culturing.

5. The method according to any one of claims 1 to 4, wherein the expression vector comprising a nucleic acid sequence encoding SEQ ID NO:4 comprises a nucleic acid sequence encoding the leader peptide of SEQ ID NO:

1.

6. The method according to any one of claims 1 to 5, wherein the expression vector comprising the nucleic acid sequence encoding SEQ ID NO:4 results in less than 20 genomic insertions.

7. 1. A method for producing a composition comprising a homodimer and a multimer of said homodimer, said homodimer comprising two monomers each comprising amino acids 21-264 of SEQ ID NO:4, said multimer of said homodimer comprising at least about 80% of the total composition; The method (a) transfecting Chinese hamster ovary (CHO) cells with an expression vector comprising a nucleic acid encoding SEQ ID NO:4; (b) culturing the CHO cells from (a) in a bioreactor using ActiCHO P medium or Acti Pro medium at a growth temperature of 37° C.±1° C.; (c) feeding the culture from (b) daily with ActiCHO Food A and ActiCHO Food B, or HyClone Cell Boost 7a and HyClone Cell Boost 7b, at a growth temperature of 37° C.±1° C. until the cells reach a density of about 5 million to about 30 million cells / mL; (d) shifting the growth temperature from 37° C.±1° C. to 32.5° C.±1° C.; (e) harvesting the homodimer and multimers of the homodimer from the culture medium; The method, wherein >30% of said multimers are higher order multimers.

8. The method described in claim 7, further comprising shifting the pH of the medium from 7.1 to 7.0 on the fifth day of culture.

9. 9. The method of claim 7 or 8, wherein the final protein titer before harvest is >3g / L, >4g / L, >5g / L, >6g / L, >7g / L, >8g / L, or >9g / L.

10. 10. The method of any one of claims 7 to 9, wherein the cell viability of the CHO cells is greater than 85% on the 10th, 11th, 12th, 13th, 14th or later day of culture.

11. 11. A method for purifying and polishing homodimers produced by the method of any one of claims 1 to 10 and compositions comprising multimers of said homodimers, comprising the steps of: (a) purifying the homodimer and multimers of the homodimer from the culture supernatant of the culture medium by affinity chromatography; (b) polishing the homodimers and multimers of said homodimers by one or more of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography; A method comprising:

12. The method of claim 11 , wherein the affinity chromatography uses a MabSelect SuRe Protein A column.

13. 13. The method of claim 11 or 12, wherein purifying by affinity chromatography comprises eluting the homodimer and multimers of said homodimer from the affinity chromatography with an elution buffer comprising sodium acetate and NaCl.

14. 13. The method of claim 12, wherein the MabSelect SuRe Protein A column is regenerated with at least 0.5 M NaOH buffer.

15. 12. The method of claim 11, wherein polishing the homodimer and multimers of said homodimer comprises anion exchange flow through chromatography.

16. 16. The method of claim 15, wherein the anion exchange flow through chromatography comprises using a Q Sepharose fast flow column.

17. 12. The method of claim 11, wherein polishing the homodimers and multimers of said homodimers comprises cation exchange chromatography.

18. The method of claim 17, wherein the cation exchange chromatography comprises using a POROS XS column.

19. The method of claim 17 or 18, wherein the cation exchange chromatography comprises using a sodium acetate elution buffer.

20. 20. The method of claim 19, wherein the sodium acetate elution buffer further comprises 36.5-38.5% 1 M NaCl buffer.

21. 12. The method of claim 11, wherein polishing the homodimers and multimers of said homodimers comprises hydrophobic interaction chromatography.

22. The method of claim 21, wherein the hydrophobic interaction chromatography comprises using a Phenyl Sepharose 6 fast flow high sub-resin.

23. A method for purifying and polishing a homodimer produced by the method of any one of claims 1 to 10 and a composition comprising multimers of said homodimers, comprising: The method (a) purifying homodimers and multimers of said homodimers from a culture supernatant of said culture medium by Protein A affinity chromatography, wherein said Protein A affinity chromatography uses an alkaline-resistant resin, such as MabSelect SuRe resin, and said purification is performed with at least two wash cycles; (b) polishing the homodimer and the multimers of said homodimer by cation exchange chromatography, wherein said cation exchange chromatography uses a high capacity, high resolution resin such as POROS XS and an elution buffer, said elution buffer being a sodium acetate buffer containing 36.5-38.5% 1M NaCl buffer. (c) polishing the homodimer and multimers of said homodimer by anion exchange chromatography, wherein said anion exchange chromatography uses an anion exchange resin such as Q Sepharose fast flow resin; (d) polishing the homodimers and multimers of said homodimers by hydrophobic interaction chromatography (HIC), wherein said HIC is a Phenyl Sepharose 6 fast flow high sub-resin and is selected to isolate or remove a specific fraction of the homodimers and multimers of said homodimers in addition to polishing; 2. A method for purifying and polishing homodimers and multimers of said homodimers, comprising:

24. 24. The method of claim 23, wherein the composition has a final protein titer of >4 g / L.

Images