NZ795260A - Manufacturing Optimization of GL-2045, a Multimerizing Stradomer - Google Patents

Abstract

The present disclosure involves optimized methods for production of hiologically active proteins termed optimally manufactured stradomers. The present disclosure further provides compositions and methods useful in the treatment of diseases and conditions including autoimmune diseases, inflammatory diseases, or infectious diseases. iseases, or infectious diseases.

Description

The present disclosure involves optimized methods for production of hiologically active proteins termed lly manufactured stradomers. The present disclosure further provides compositions and methods useful in the treatment of diseases and ions including autoimmune diseases, inflammatory diseases, or infectious diseases.

NZ 795260 Manufacturing Optimization of 5, a Multimerizing Stradomer REFERENCE TO RELATED ATIONS This application is a divisional of New Zealand ation No 753108 which is the New Zealand national phase entry of , which claims priority to US Provisional Application No. 62/432,402 filed 9 December 2016. Each of these applications is herein incorporated by reference in their entireties.

FIELD OF THE ION This invention relates lly to the fields of immunology, autoimmunity, inflammation, and tumor immunology. More specifically, the present invention relates to zed methods of manufacturing GL-2045. The invention also relates to novel compositions comprising such optimally manufactured GL-2045, as well as methods of using the GL-2045 itions.

The invention further relates to treating or preventing ogical conditions such as autoimmune diseases and inflammatory diseases.

BACKGROUND OF THE INVENTION Pooled human intravenous globulin (IVIG) has been used since the early 1950’s to treat immune deficiency disorders and, in more recent decades, autoimmune and inflammatory diseases. IVIG mediates tolerogenic immune effects via several mechanisms including binding of IVIG aggregates to complement C1q and Fc gamma receptors (FcγRs) and cross-linking 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 ation of sterile, purified immunoglobulin G (IgG) products manufactured from pooled human plasma that typically contains more than 90% unmodified IgG, with small and variable amounts of the multimeric immunoglobulins, IgA or IgM (Rutter A et al., J Am Acad Dermatol, 2001, Jun; 44(6): 1010-1024).

[Annotation] yzus [Annotation] yzus {8995} Substantial published data t that the small, ated lgG fraction of lilVlG, specifically the Fc portion of those aggregates, is disproportionately effective in the treatment of certain diseases mediated by patliologic immune complexes. it has been observed that traces (la 59/15) of lgG are present as multinieric forms within lVlG, and lgG dimers can make up 5—15% of hl‘v'lG. atives to lVlG therapy using recornhinantly—produced Fc multimers that avidly bind Ft: Receptors and complement component Clq, similar to lVlG aggregates, have been described (See US Patent Application Publication Nos. 2010/0239633, US l 56765, US 2015/02l8236, and PCT Publication No. W0 ZOl 53/132364). {9996} One such Fc multimer, Gil—2045, has been previously disclosed (US Patent Application Publication No. ZOE/0156765). GLnZOAlS is a multimerizing general stradonier that is a recombinant mimetic of lVlG. GL—ZO45 binds most or all of the ligands to which inimunoglohulin lgGl Fc binds, Further, GL~2045 binds with high affinity and avidity to all canonical receptors and to complement Clq, and has a 10 - 1,000 fold greater in wire efficacy compared to lVlG. onally, {ill-2045, or its murine equivalent, is ett‘ective in numerous animal models of autoimmune disease including en~induced arthritis, mental autoimmune neuropathy, idiopathic thrombocytopenic a, and experimental autoimmune myasthenia gravis As such, (ill—2045 also has potential clinical utility in treating a wide range of autoimmune diseases, including but not limited to thic thromhocytopenic purpura, chronic inflammatory polyneuropathy, inultil‘ocal motor neuropathy, niyastl'ienia gravis, organ transplantation, and rheumatoid arthritis, {8807] ln addition to the advantage of Gil—2045 over lVlG in potency and efficacy, Gd...~ 2045 demonstrates several advantages in the manufacturing process. lVlG is pooled human blood product, meaning that it is derived from the blood of tens of thousands of donors whose serum is then mixed together and subsequently ed to remove viruses and other infectious agents, as well as ated lgG. As such, access and supply are limited and production costs are high.

Additionally, there is a significant degree of variability between lots of lVlG. Conversely, GLu 2045 is reconihinantly produced and therefore obviates the difficulties of supply and production costs while providing greater control over the manufacturing process. {0098} The (EL-2045 mer binds with ty and without substantial avidity to Fe s including E'c gamma receptors and ment Clo. it also naturally forms higher order multimers capable of binding to canonical receptors with avidity. it is these higher—order multimers [Annotation] yzus [Annotation] yzus of GLuZOA'lS that mimic the ed efficacy of the eric fractions ofWK}. Standard cell culture conditions, r, produce varying levels of cell viability, degrees of multimerized proteins, and protein titers. Therefore, there is a need in the art for methods of cturing GLu 2045 that results in a defined multimer pattern, and particularly one that results in an increased tage of highernorder multimers while optimizing cell viability and protein titer.

SUMMARY BF THE INVENTION {9999} The present ion provides for all three of improved cell viability, improved high protein titer, and a sing and substantial increase in the percentage of higher—order multimers relative to standard manufacturing techniques. This optimized manufacturing method therefore, provides for optimally manufactured. GL—Ztl45 compositions with enhanced efficacy for treating matory diseases as compared with non~optiinally manufactured 45 compositions. Optimized manufacturing of GLnZOZlS es optimized upstream manufacturing methods and, in some embodiments optimized downstream methods. Optimized upstream manufacturing methods a) generate high protein titers, h) maintain high cell viability to minimize cell debris, and c) retain. both the highly d mu ltimers of the homodimer that are essential for the functioning of Gl_.-—2045 and, it‘desired, the horn odimer. Optimized downstream manufacturing methods include various purification techniques that are employed specifically to in a selected multimer profile of Gl..—2045. Thus, in some embodiments, provided herein are GL~2045 compositions with a d multimer profile {0919} In some embodiments, a method for producing GL~2045 is provided comprising culturing Chinese l-lamster Ovary (Cl-l0) cells that have been stably ected with an expression vector encoding (EL—2045 at 37°C ti:- l°C until the CHO cells reach a cell density of about 5 to about million cells/rnl...; shifting the growth ature from 37%: -:l:. l"C to 326°C :_l-_- loC; and harvesting (EL-2045 from the culture media, In some embodiments, the cells are grown to a density of about l0 to about 25 million cells/ml, prior to the shifting growth temperature. in some embodiments, the cells are grown to a density of about 10 to about l 5 million cells/nil, prior to the ng growth ature. In some embodiments, the cells are grown to a density of about l5 to about 20 million cells/mL prior to the shifting growth temperature. in some embodiments, a dual temperature shift is employed with a shift from 37°C :: PC to 34°C :i: l"C on about day 3 with a second temperature shift from 34°C :l: 10C to 31°C :i: 1°C on about day 7 of hioreactor culture.

[Annotation] yzus ation] yzus lllllll} In some embodiments, the } cells are cultured in in ActiCIIO I) base culture media, In some embodiments, the {II-IO cells are fed during culture with ActiC’I—IO feed A and ActiCI-lt) feed B. In some embodiments, the (II—IO cells are fed every other day. In some embodiments, the expression vector encoding (ids—2045 comprises the leader peptide of SEQ ID NO: 1. In some embodiments, the expression vector ng (EL—2945 further ses a piggyBac transposase recognition sequence and is transfected with a vector encoding a piggyBac transposase. In some embodiments, the expression vector ng GL—2045 results in fewer than genomic ions. {9912} In some embodiments, a recombinantly produced. 5 made by the methods described herein is provided. In some embodiments, an expression vector is provided ng GL—204’5 comprising a GIL-2945 expression cassette, wherein the (EL—2045 expression cassette is flanked by piggyBac minimal inverted repeat ts. {$013} In some embodiments, a method for producing (EL—2045 is provided comprising transfecting CHO cells with an expression vector described herein, culturing the CHO cells in a ctor with ActiCHO P media. at a growth temperature of 37° C i l0, feeding the cultures of CHO cells with Acti CHO Feed A and Acti CHO Feed B daily at a growth temperature of 37° C :l: PC until the cultures reach a cell density of about l0 million to about 15 million cells/trill, shifting the growth temperature from 37“ C :1: 1°C to 325°C :: 1°C, and harvesting (IL~2045 from the culture media, wherein the methods result in a cell viability of >80% at Day 21, and a final protein titer of > 9,000 mg/mL of which >70% of GIL—2945 is present as a multimer, wherein >30% of the rnultimers are higher order multimers Gil—2045. In some embodiments, the cell viability exceeds 95% at day l 8 of culture. In some erribodiments, the percent of in ultimers exceeds {MM} In some embodiments, a method of purifying Gin-2045 produced by the methods described herein is provided comprising purilying (IL-2045 from the culture supernatant by affinity chromatography and polishing (ill-2045 by one or more of cation exchange chromatography, anion exchange chromatography, and hydrophobic ction chromatography. {9915} In some embodiments, depth filtration is employed prior to affinity chromatography. In some embodiments, the depth filter is the XOI—IC (Millipore). In some embodiments, the depth filtration unit removes a high tage of DNA from supernatant. In some embodiments that depth filtration unit is Emphaze’l‘M AEX Hybrid Purifier (3 M).

[Annotation] yzus [Annotation] yzus {9916} In some embodiments, the affinity chromatography uses a protein A coiumn. In some embodiments, the protein A column ses an Naifl-I—resistant resin. In some embodiments, the protein A resin is a MabSelect Sulie resin. In some embodiments, cation by affinity chromatography comprises utilizing one of three different wash buffers to optimize purification conditions. In some embodiments, purification by affinity chromatography comprises eiuting (IL—2045 from the affinity tography column. In some ments, eiuting GL' 2045 comprises elution with a pH gradient. In some embodiments, g GL~2045 ses n without a pH gradient. In one embodiment, elution is performed using a glycine buffer. In another ment, elution is performed using an acetic acid buffer. In some embodiments, the affinity chromatography column is regenerated to remove bound {EL—2645. In some embodiments, the affinity tography column is rated more ntly than suggested by the manufacturer. In some embodiments, the affinity chromatography column is regenerated prior to each purification cycle. In some ments, the affinity chromatography column is regenerated with a O. 5 M NaOH buffer. {(3917} In some ments, polishing GI_.—2G45 comprises anion exchange flow through chromatography. In some embodiments, anion exchange flow through. chromatography comprises using a. Q Sepharose Fast Flow column In some embodiments, polishing GI_,—2045 comprises cation. exchange chromatography. In. some embodiments, cation exchange chromatography comprises using a PQROS XS column In some embodiments, cation exchange chromatography comprises using a sodium acetate elution buffer In some embodiments, the elution buffer further comprises 365—39094) of a 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, polishing GL— 2045 comprises hydrophobic interaction chromatography. In some embodiments, hydrophobic interaction chromatography comprises using a Butyi FIT resin. In some embodiments, hydrophobic interaction chromatography comprises using a Phenyi I-II’ resin. In some embodiments, hobic interaction chromatography (“I-II 3”) comprises using a I’henyl Sepharose 6 Fast Flow I-Iigh Sub resin. In one embodiment, the I-IIC method is in flow through mode and in another embodiment the I-IIC method is in binding mode. In some embodiments, the I-IIC resin results in isolation of the (IL-2045 homodimer. In some ments, hydrophobic interaction chromatography comprises using an Octyi FF resin. In some embodiments, the column results in the removal of un—ordered aggregates of (EL—2045.

[Annotation] yzus [Annotation] yzus {9913} In some embodiments, a method for purifying (EL—2045 is provided comprising purifying (Eleni-2045 from the culture supernatant by protein A affinity chromatography, n the protein A column uses an alkaline—resistant medium such as the MabSelect SuRe medium, wherein the purification is performed with at least two wash cycles, and wherein clean in place (CIP) procedures are performed after each purification run with a high NaOH regeneration step such as (‘5. 5 M NaOH buffer. {$019} In some embodiments, a . for ing (EL—2045 is provided comprising polishing {EL—2045 by cation exchange chromatography, wherein the cation exchange column contains a high—capacity, high—resolution resin such as POROS KS and. wherein the elution buffer is a sodium acetate buffer comprised of 36.569096 of a l M NaCl buffer. In some embodiments, the method for ing 45 further comprises polishing (EL—20.45 by anion exchange chromatography, wherein the anion ge column ns a strong anion exchange medium that has high chemical stability, allowed clean—in—place and sanitation protocols, such as the Q Sepharose Fast Flow , In some embodiments, the method for purifying Gl_,—2045 further comprises polishing GL~2045 by hydrophobic interaction chromatography, wherein the hohic interaction medium. is a Butyl FF, a Phenyl HP, or an Octyl. FF resin and is selected to isolate or remove a particular fraction of (EL—2045 in addition to polishing. In some embodiments, the method for purifying and/or polishing GI.,~2045 results in a final protein titer of GLQOL’ES > 4 g/L after all filtration and chromatography steps (ie the final Drug Substance). In some embodiments, the final protein ition of (IL—2045 comprises >70% multimers. In some embodiments, >a8% of the multimers are higher order multimers as analyzed by analytical SEC-HPLC {8826} In some embodiments, a purified {1114-2045 made by the methods described herein is provided. in some embodiments, the ed GL~”O45 made by the methods described herein has a d multimer pattern that minimizes the percentage of mers and/or dimers of the homodimers, or otherwise balances the tage of homodimers, lower order multimers, higher order m‘ultimers, and t order multimers. In some embodiments, a method of treating or preventing an inflammatory, autoimmune or infections disease or disorder in a suhject in need thereof with the recombinantly produced, purified (EL-2045 described herein is provided. In some embodiments, the disease or er is selected from idiopathic thrombocytopenic purpura, c inflammatory polyneuropathy, multifoeal motor neuropathy, myasthenia gravis, organ [Annotation] yzus [Annotation] yzus transplantation, and rheumatoid arthritis. In some embodiments, the 45 is administered intravenousiy, subcutaneously, orally, intraperitoneaiiy, guaiiy, bucaiiy, transdermaiiy, via subdermai implant or intramuscuiariy. 1} in some embodiments, a recombinantiy ed GL~2045 composition is provided, wherein the homodimer fraction of the Gig—2045 composition ses iess than about % of the totai composition. In some embodiments, the homodimer fraction comprises 12-19% of the total composition. In other embodiments, the homodimer fraction comprises Md 9% of the total composition. In some embodiments, the homodimer fraction comprises 155—17594; of the total composition. In another embodiments, the homodimer fraction comprises about 16.2% of the total composition. {9022} In some embodiments, a recombinantiy ed (EL—2045 composition is provided wherein the dimer of the homodimer fraction of the GL—‘ZO45 composition comprises about 7% to about 12% of the totai composition. In some ments, the dimer of the homodimer fraction comprises about 9% to about 11% of the totai composition. in other embodiments, the dimer of the homodimer fraction comprises about 10% of the totai composition, {9023} In some embodiments, a recombinantiy produced GL-‘ZO45 composition is provided, wherein the trimer of the homodimer fraction of the GL~2045 composition comprises about 5.5% to about 11% of the total composition. In some embodiments, the trimer of the homodimer fraction comprises about 6.5% to about 8% of the totai composition. in other embodiments, the timer of the homodimer on comprises about 7% of the total composition. {(3824} in some embodiments, a recombinantiy ed 045 composition is provided, wherein the tetramer of the homodimer fraction of the (EL-2045 composition comprises about 10% to about 16% of the total ition, in some embodiments, the tetramer of the homodimer fraction comprises about 13% to about 15% of the total composition. In other embodiments, the tetramer of the homodimer fraction comprises about 14% of the totai composition. {(3025} in some embodiments, a recombinantiy produced GL~”O45 composition is provided wherein the pentamer of the mer fraction of the GLQMS composition comprises about 6% to about 9% of the total composition. in some embodiments, the er of the homodimer fraction ses about 7% to about 8% of the totai composition. in other ation] yzus [Annotation] yzus embodiments, the pentamer of the homodimer on comprises about 7% of the total composition. {9926} In some embodiments, a recombinantiy produced Gil—2045 composition is provided, wherein the hexamer of the homodimer fraction of the GLnZOétS composition ses about 10% to about 14% of the total composition. In some embodiments, the hexamer of the homodimer fraction comprises about 12% to about I’ % of the total composition. In other embodiments, the hexamer of the homodiiner fraction comprises about 12.7% of the total composition. {9927} In some embodiments, a recombinantiy produced (EL—2045 composition is ed wherein the highest order multimers (he. , those in the 7nmer of the homodimer and above fractions) comprise at ieast about 28% of the totai composition. In some embodiments, the t order muitimers comprise no more than 35% of the totai composition. In some ments, the highest order er fractions comprise from about 30% to about 34% of the total composition, In other embodiments, the highest order multimer fractions comprise about 31.4% of the total composition. {9028} In some embodiments, a recombinantiy produced GL-‘ZO45 composition is provided wherein (a) the homodimeric fraction comprises less than about 20% of the total composition; (h) the highest order muitimer fractions comprise at least about 28% of the total composition; (at) the dimer of the homodimer fraction comprises from about 7% to about 12.5% of the total composition; (d) the trimer of the homodimer fraction comprises from about 55% to about 11% of the totai composition; (e) the tetramer of the homodimer fraction comprises from about 30% to about 16% of the totai composition; (f) the er of the homodimer fraction comprises from about 6% to about It % of the totai composition; (g) the hexamer of the homodimer fraction comprises from about 10% to about {4%) of the total fraction; [Annotation] yzus [Annotation] yzus (h) the dimer of the iner through hexamer of the homodimer fraction comprises from about 40% to about 60% of the total composition, (iv) the trimer of the homodimer through the hexainer of the homodimer fractions comprise from about 32% to about 50% of the totai composition; {j} the tetramer of the homodimer through the hexamer of the homodimer fraction comprise from about 26% to about 39% of the totai composition; (It) the pentamer of the homodimer through the hexamer of the homodimer fraction comprise from about 18% to about 23% of the totai ition; or (I) any combination of (rd—(k). {9029} In some embodiments, a recombinantiy produced GIL-2045 composition is provided, wherein approximately 80% of the total composition comprises higher order multimers, meaning the dimer of the homodiiner and above (to, band 2 and above). In some embodiments, approximately 60—80% of the totaI recombinantiy ed GLMZO/ifi composition comprises the trimer of the homodimer and above (to, hand 3 and above). In some embodiments, about 54~72% of the total of the recombinantly produced Git-20115 composition comprises the tetremer and above (ten, band 4 and above), In some embodiments, a GIpZOr-ifi is provided wherein approximately 4 57% of the total composition comprises the pentamer and above (1'. or, band 5 and above). In some embodiments, about 38—51% of the totaI of the recombinantly produced GInZOI-ES composition comprises the hexamer and above (128., band 6 and above} {3039} In some ments, a inantly produced (Hi—2945 is provided wherein bands 2~6 of the composition (ie the dimer of the homodirner through the hexamer of the homodimer) comprise about 39—61% of the composition. In some ments, a recombinantiy produced GI_,-2045 is provided, wherein bends 3—6 of the composition (i.€., the trimer of the homodimer through the hexamer of the homodimer) comprises about 325094: of the ition.

In some embodiments, a inantiy produced (EL-2045 is provided wherein bands 4-6 of the composition (Li-3., the tetramer of the homodimer h the hexamer of the homodimer) comprises about 26—39% of the composition. In some embodiments, a recombinantiy produced 45 is provided wherein bands 5nd of the ition (Le, the er of the homodimer through the hexamer of the homodimer) comprises about 16—23% of the composition.

BRIEF DESKITRII’TION 01“ THE DRA‘WINGS [Annotation] yzus [Annotation] yzus {8931} FIG. lA u FlG. lB illustrate GLu2045 fractionation by size exclusion chromatography (FIG; 1A) and analysis of the resulting ons by non—reducing gels (). {9932} Fl G. '2 illustrates biolayer interferometry analysis of GLuZQL'lS fractions. {8633] _ FIG. BB illustrate gel analysis (FIG; 3A) and size—exclusion fractionation results (FlG. SE) for GL—ZIMS. {9034] — ) illustrate the effects of GLnZOrtS fractions in a complement— dependent cell killing assay. {9035} n illustrate an elution clii'oniatcgrain () and ge analysis of GL~2045 for use in an FcyRIlIa binding assay (FIG. SB and 5C). {8936} u illustrate the g of eluted fractions shown in FIG 5 to la () and the bestufit curve (). {(3837} rates FEDS—Page analysis of anion exchange fractions: GI...~Gl_ilVl—Ol:= recombinant, unfractionated Fc (GOOD, GL-Glrlyl-GZ unfractiona‘ted GL-ZMS, GL— ILl‘vl-OS onated (Ila—2.04:3 at pH 6.0, Gl..—Gl.il_\/I—06 fractionated GL-2045 at pl-l 6.5, GI.,-GLM—O7 fractionated 045 at pI-l 7.0, GL—GLl‘vLOS fractionated GL-2045 at pI-l 7.5. {(3338} illustrates the results of a neutrophil chemotaxis assay with CSa as the chemoattraclant in the presence of unfractionated or fractionated Gl.i~2045: GL—GLli/LOl recombinant, tionated Fc (GOOl), GL—GLM-O'Z unfractionated (IL-2045, GL—GLM-OS fractionated GLu2045 at pH 6.0, GL—GLM-Oo fractionated (EL—2045 at pH 6.5, GL—GLM—O? fractionated (EL—2045 at pl-I 7,0, GL~GLM—08 fractionated (EL—2045 at till 7. 5. {9939} illustrates cell y 1 in millions/niL) of CHO cells grown in a panel of different media on days 4, 8, and 10 of culture. {864%} FIG. l0 illustrates cell Viability (0/6)) of CHO cells grown in a panel of different media on days 4. 8, and 10 of culture. {8041} FIG. ll rates protein titer (mg/niL) of CHO cells grown in a panel of different media on day ll) of culture. {@942} FIG IZA - B illustrate gel analyses of GL—“045 protein ed from CHO cells grown in the panel of media rated in FIGS. 94 l. {9043} FlG l3A — B illustrate feeding schedules for I’ewerCI—IO3 CD, ADCF—Mab ne, and ActiCHO F media (FIG. BA), and feeding schedules for Cellvento, BalanCI} CEO Growth A, Cl.) FortiCHO Life, and CD4MCHO Hyclone media. (38).

[Annotation] yzus [Annotation] yzus {8944} FIG. l4 illustrates cell density (e6 cells/inL) of (II-l0 cells grown in a panel orn ent media + feed combinations on days O—ll of culture {@945} illustrates cell viability ) of CHO cells grown in a panel of different media + feed combinations on days Owl l of culture. {6046} illustrates protein titer (nig/rnh) of (EL—2945 front CHO cells grown in a panel of different media + feed combinations on days 0—11 of culture. {3047} illustrates SDSJ’AGE analysis of the effects of the media + feed combinations and schedules illustrated in A—13B on GLnZOrlS inultirnerization. {9948} A — 1) illustrate the effects of O-P media + feeding everyday (Red), and ActiCHO—F media + feeding every other day (Blue) on cell density (FIG. ltlA), cell viability (3), culture pH (C), and Gila-“O45 n titer (FIG. ISD). {@949} FIG. l9 rates the effect of ActiCHO—F media + feeding everyday (Red) and ActiCHO—P media + feeding every 3 days (Blue) on G_i~2045 n titers. {0956} FIG, 20A ~ FIG ZOE illustrate the effects of ing AtttiCHO Feed A with FowerCHO‘Z (Red) base media on cell viability (A) and (EL-2045 protein titer (B) compared to using ActiCHO Feed A. with ActiCHO—F base media (Blue).

{MESH A — C illustrate the effects of optimized shake flask conditions on cell density (FIG. ZlA), cell viability (FIG: ZIB), and GL—ZG45 protein titer (FIG. ZIC). {8852} FIG 22 illustrates SIDS-PAGE analysis of protein A purified Glm2045. {9053} A - FIG; .438 illustrate an elution profile from protein A column after elution of (EL—2045 by pl-l gradient elution (A) and SIDS—PAGE analysis of isolated fractions (B) {(3854} rates an elution tograni and non—reducing SEES—PAGE analysis. {6055} illustrates an elution chroniatograni and non—reducing SIDS—PAGE analysis. {(3056} illustrates an elution chromatograrn ofRun Cl ~C3. {8957} illustrates non—reducing EDS—PAGE analysis of elation peaks 3896—3996 from run C1 {13. {8953} illustrates ometry analysis of ion chromatography purified GL_2045.

[Annotation] yzus [Annotation] yzus {8959} Fl G. 29A _ EEG. 29B illustrate elution profiles from l-llC columns (upper ) and SDSuPAGE is of elution and flowntlirough fractions (F'1‘) (lower panels). {@960} illustrates an elution profile from HR: column polishing of M045 (right panel) and NuulKAGE analysis (right panel). {6061} HQ. 3l illustrates an elution profile from BIC column polishing of ill/£045, {0062] illustrates an elution profile from Hill: column polishing ofMOM and SEE“ PAGE analysis. {9063} illustrates a defined niultimer pattern of an optimally manufactured Glam 2045 composition.

DETAILED DESCRIPTION OF THE EGN {9964] The approach to production of optimized recombinant (ills-2045 described herein includes optimized upstream manufacturing s that result in ed Gl_,-2045 rnultimerization while optimizing cell viability and protein titer. in some embodiments, the optimized state is carried through to drug substance by optimized downstream manufacturing.

Further, provided. herein. are compositions sing Gil-2045 with a defined niultimer n.

The itions provided herein have utility for treating autoimmune disease, inflammatory disease, allergy, antibody—mediated disease, and ccmplement~mediated disease. {@865} As used herein, “drug substance” refers to the final dosage form of GL-ZOL’ES as sold by the cturer. {8866] As used herein, the use of the word “a" or “an" when used in conjunction with the term "comprising" in the claims and/or the specification may mean ”one,” but it is also consistent with the meaning of ”one or more," "at least one," and ”one or more than one." {8967} As used herein, the terms "hiomimetic", "biomirnetic molecule", ”biomimetic compound", and related terms refer to a human made compound that imitates the function of r compound, such as pooled human Intravenous globulin (“hIVlG”), a monoclonal antibody or the Po fragment of an antibody. ”Biologically active" liiomimetics are compounds which possess biological activities that are the same as or similar to their naturally occurring counterparts. By ally ing” is meant a molecule or portion thereof that is normally found in an organism. By naturally occurring is also meant substantially naturally occurring. "immunologically active” hiomimetics are biomimetics which exhibit immunological activity the ation] yzus ation] yzus same as or similar to naturally occurring immunologically active molecules, such as antibodies, cytol<ines, interleukins and other immunological molecules known in the art. ln preferred embodiments, the biomimetics of the t invention are optimized multimerized stradomers, as defined herein (6g. optimallyl manufactured (EL—2045). {9068} By “directly linked” is meant two sequences connected to each other without intervening or extraneous sequences, for example, amino acid sequences derived, from insertion of restriction enzyme recognition sites in the DNA or cloning fragments. One of ordinary skill in the art will understand that “directly linked” encompasses the on or removal of amino acids so long as the multimerization capacity is substantially unaffected. {9069} By “homologous” is meant identity over the entire sequence of a given nucleic acid or amino acid sequence. For example, by “80% homologous” is meant that a given sequence shares about 80% identity with the claimed sequence and can include insertions“, deletions. substitutions, and frame . One of ordinary skill in the art will understand that sequence alignments can he done to take into account insertions and ons to determine identity over the entire length of a sequence. {9079} It has been bed that hlVlG binds to and fully saturates the neonatal Fe receptor (PcRn) and that such competitive inhibition of EcRn may play an important role in the biological activity of lilVIG (cg. F. lin at all, Human logm 200.5, 66(zl-)4lr03~410). Since innnunoglohulins that bind strongly to Fey receptors also hind at least to some degree to FcRn, a skilled n will recognize that stradomers capable ofhinding to more than one Fey receptor will also bind to and may fully saturate the FcRn. {(13071} There are two human polymor‘phs of lgGl termed DEL and REM polymorphs. The DEL polymorph has a D at position 356 and an L at position 358; the EEM polymorph has an E at on 356 and an M at position 358 (Kahat numbering, SEQ ll) N’Os: 2 and 3, EEM and DEL polyinorphs, respectively). The mers provided herein may comprise either the DEL or the EEM lgGl polymorph. Thus, even if a ce for a particular mutant is explicitly produced in the context of the DEL polymorphism; one of skill in the art will understand that the same ons may be made to the EEM polymorph to yield the same results. {9072} US 239633 discloses using linked immunoglobulin Fe domains to create orderly multimerized iinrnunoglohulin Fe bioniirnetics of hlVlG (biologically active ordered niultiiners known as stradomers), which include short sequences including restriction sites and [Annotation] yzus [Annotation] yzus affinity tags between individual components of the stradomer, for the treatment of pathological conditions including autoimmune diseases and other inflammatory conditions. See US 2010/0239633, incorporated by reference in its entirety. US 20l3/0l 56765 discloses mers wherein the dual components are directly , rather than separated by restriction sites or affinity tags. US 20l3/Ol56765 also specifically discloses a multimerizing stradomer ((39—2045) comprising an lgGch domain with an lgGZ hinge multimerization domain directly linlred to its C—terminus, which exhibits enhanced multimerization and ment binding ve to the Na terminal linked construct (erg, GLUZOU), described in US ZONE/0239633). See US 20l3/0l56765, incorporated by reference in its entirety. The structure of GL-ZO45 is: IgGl Hinge — lgGl {Tl-'12 lgGl CH3 — lgGZ Hinge and (EL-"2045 is provided as SEQ ll) NO: 4 and 5 (HEM and DEL polymorphs, respectively).

Srmdamer Urdulfonomer {9973} As used herein, the term ”stradomer unit monomer” refers to a. single, uous e molecule that when associated with at least a second stradomer unit monomer, forms a homodimeric “stradorner unit” comprising at least one Fe domain, and in the case of Gil-2045 an lgG‘Z hinge multimerizati on domain. In preferred embodiments, stradomer units of 45 are sed of two associated mer unit monomers. However, a GL-Zt'fllfi stradoiner may also contain three or more stradom er unit monomers, {0074} The optimally manufactured stradomer of the current invention (optimally manufactured (EL—2045) contains a direct linkage between the N—terminus of the lgGl Fc r and the C us of a leader peptide (SEQ ID NO: l) and the C terminus of the l'gGl Fe and the N us of the multimerization domain lgGZ hinge (SEQ ll) N0: 6) {8975} As a clarifying example, the skilled artisan will understand that the optimally manufactured stradonier molecules of the present invention may be constructed by preparing a polynucleotide molecule that encodes an F0 domain monomer and a multimerizing region. Such a polynucleotide molecule may be inserted into an expression vector, which can be used to orm a population of bacteria or transfect a population of mammalian cells. Stradomer unit monomers can then be produced by culturing the transformed bacteria or transfected mammalian cells under appropriate e conditions. For example, a clonal cell line continuing a pool of stably transfected cells can he achieved by ing cells with genetecin’GrllS. Alternatively, cells can [Annotation] yzus [Annotation] yzus be transiently transfected with DNA encoding the optimally manufactured stradomer of the current invention (cg DNA encoding the stradomer according to SEQ ll) N0: 4 or 5:) under the l of the CMV promoter. The expressed stradomer unit monomers can then form functional stradomer units and stradorners upon either self—aggregation of the stradomer monomers or units or association of stradomer monomers using stradomer monomer linkages. The expressed stradomei's can then be purified from the cell culture media by downstream manufacturing methods described herein (cg, affinity chromatography, ionwexchange chromatography, and/or hydrophobic interaction chromatography). One of skill in the art will understand that the leader peptide included. in the c acid construct is used only to facilitate production of the stradomer unit monomer peptides and is cleaved upon expression ofthe mature protein. Thus, the biologically active biomimetics of the t ion do not comprise a leader peptide. r Srradomer {9976} In one embodiment, the optimally manufactured Gil—2045 made in ance with the t disclosure is a cluster stradomer. A ”cluster stradomer" is a etic that has a radial form with. a central moiety "head” and two or more ? wherein each leg comprises one or more Fc domains that is capable ol‘hinding at least one Fc gamma receptor and/or complement. A cluster stradomer is also known as a. “multimerizing stradomer” by virtue of the ce of a multimerization domain that results in rnultimerization of the stradcmer. Thus, serial stradcmers which contain multiple Fr: domains on one stradorner r molecule may still be classified as a cluster stradomer or rnultimerizing stradomer so long as the molecule also contains at least one multimerization domain. Each cluster stradomer is sed of more than one homodimeric protein, each called a "cluster stradoiner unit " Each cluster stradomer unit is sed of at least one region that multimerizes and a "leg region that comprises at least one functional Fc domain.

The multimerizing region creates a cluster stradomer ”head” once multimerized with another cluster stradomer unit. The leg region may be capable of binding as many complement molecules as there are Fc domains in each leg . For example, the leg region may bind as many Clq molecules as there are Fc domains in each leg region. Thus a cluster stradorner is a hiomiinetic compound capahle of binding two or more Cl q molecules, thus preventing complement-mediated lysis also known as Complement Dependent Cytotoxicity (CDC).

[Annotation] yzus [Annotation] yzus {0077} The nerizing region contained within the optimally manufactured stradomer of the current invention is the lgGZ hinge region. As is known in the art, the hinge region of human lgGZ can form covalent dimers (Yoo, EM. er a}. J. linmunol. l70, 3l34-3l38 (2003:); Salfeld Nature h. 25, l369~l372 (2007)}. The dimer formation of lgGZ is potentially mediated through the lgGZ hinge structure hy CUC bonds (Yoo er a] 2003), suggesting that the hinge structure alone can mediate dimer ion. The amount of lgGZ dimers found, in human serum, however, is limited. it is estimated that the amount of lgGZZ existing as a dimer of the homodimer is less than l0% of the total lgGZ (You or a]. 2003). Furthermore, there is no quantitative evidence of the multimerization of IgG2 beyond the dimer of the homodimer. (Yoo er of. 2003). That is, native lgGZ has not been found to form higher order rnultimers in human serum. The lgGZ hingem ning stradomers tag, optimally manufactured {EL—“045) are present as higher-order multimers and, unlike native lgGZ in human serum in which the lgGZ hinge interactions are variable and dynamic, Sis—2045 has been trated to form highly stable multiniers evidenced on non~reducing SDS—PAGE gels, analytical ultracentrifugation, and 3—month stability s at l00% humidity at 37° C. Furthermore, it is also surprising that the amount of multiniers in the lgGZ hinge-containing stradornet preparations are significantly higher than the approximately l0% of dimers and no ners observed for IgGZ in human serum. For example, the percent of stradomets that are multimers, including dimers, s, tetraniers and higher order niultiniers of the homodimer exceeds 20% and may exceed 30%, 40%, 50%, 60%, 70%, 80%, or even 90%. in an especially preferred embodiment, the percent of Gil—2045 present as a homodimer is n l 0 and 20% and the corresponding percent of Gil-2045 t as highly ordered multimers oi" the hornodimer is greater than 70%, {(3878} The amino acid sequence (EL—2045 is described in SFQ ll) NO: 4 and 5. {0079} The term ”isolated" polypeptide or peptide as used herein refers to a polypeptide or a peptide which either has no naturally—occurring counterpart or has been separated or purified from components which naturally accompany it, egg, in tissues such as pancreas, liver, spleen, ovary, , muscle, joint tissue, neural , intestinal tissue, or breast tissue or tumor tissue (6.3, hreast cancer tissue}, or body fluids such as blood, serum, or urine. ’l‘ypically, the polypeptide or peptide is considered "isolated” when it is at least 709/5, by dry weight, free from the proteins and other naturallyuocourring organic molecules with which it is naturally associated.

Preferably, a ation of a polypeptide (or peptide) of the invention is at least 80%, more [Annotation] yzus [Annotation] yzus preferably at least 90%, and most preferably at least 99%, by dry weight, the ptide (peptide) of the invention. Since a polypeptide or e that is chemically synthesized is inherently separated from the components that naturally accompany it, the synthetic polypeptide or peptide is “isolated.” {9086} An isolated polypeptide (or peptide) of the invention can he obtained, for example, by expression of a recombinant nucleic acid encoding the polypeptide or peptide or by chemical sis. A polypeptide or peptide that is produced in a cellular system ent from the source from which it naturally originates is ”isolated” because it will necessarily be free of components which naturally accompany it, In a red embodiment, the isolated polypeptide of the current invention contains only the sequences corresponding to the lgGl lE'c monomer and the lgGZ hinge multimerization domain (SEQ ID NO: 6), and no further sequences that may aid in the cloning or purification of the protein (ag, introduced restriction enzyme recognition sites or purification tags). The degree of isolation or purity can be measured by any appropriate method, cg, column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis. [lsl’omtfact‘uring diet}:(Iris lllllSl] 045 forms ordered inultimers of the honiodimer and is active in the homodimer and all of the er fractions It is critical. to GL~2045 function that the cturing processes result in an zed multimer profile. As used herein, “optimized multimer profile” or “optimized multirnerization profile” refers to the nati on of homodirners and highly ordered multimers of (EL—2045 that results in the desired biological outcome for GL‘“ 2045 as an lVlG mimetic leg, enhanced binding to Clq with initial activation of the complement system, and/or subsequent tion of complement activation and prevention of CDC, for example without being limited by theory, at the level of Ell/(33b). A skilled artisan will recognize that it may be advantageous to isolate s multiiner ons from the optimally manufactured (EL—2045 as a separate product, either alone or combined with other elements, including for other therapeutic purposes. For example, as provide in the Examples, the larger multimer fractions of (EL—2045 are more active than smaller ner fractions in binding to Clo and modulating downstream complernentsincdiated effector function and at binding low affinity chRs. The methods of the present invention are thus directed to not only 2045 compositions comprising the optimized multimer profile, but. also to (31.1-2045 compositions comprising only select [Annotation] yzus [Annotation] yzus multimers based on the desired effector function. in such embodiments, the optimized multimer profile of GL~2045 that results in one desired biological outcome may differ from the optimized multimer profile that s in another desired biological outcome. {8682] Without being bound by theory, it is thought that the homodimer serves as a receptor and ligand buffer, similar to unaggregated lgGl. The higher order niultimers bind with increasing avidity to low ty For receptors and to complement factors (erg. Clq, which is hexameric) and, as described herein, demonstrate enhanced biological efficacy compared to diniers or lower order ers (cg, dimers, trimers, and/or tetramers of the GL~2045 homodimer} Therefore, the degree of multimerization is a critical am and downstream manufacturing consideration in the production of clinically efficacious Git-2045. As such, it is not only desirable to in optimal cell ity, high protein titer, and optimal multinierization profiles of or...—2o45 through optimized upstream manufacturing methods, but also to maintain and/or enhance l multimerization es of (EL—2045 through optimized downstream manufacturing methods. {$983} in some embodiments, optimized manufacturing methods bed herein result in. a Gil—2045 protein composition in which at least 70% or at least 80% of Git—2045 is present as non—honiodimers (ewg dimers of the hornodinier, trimers of the homodinter, etc). in some embodiments, greater than 70% or greater than 80% of GL~2045 is present as nonvhomodiniers For example, optimized manufacturing methods may result in a Ski/1045 protein composition wherein 80%, 85%, 90%, 95%, or greater of the (EL—"7045 is present as non‘hornodimers. In some embodiments, the protein composition comprises at least 28% or at least 30% of (EL—2.045 present as the highest order multimers (2e. 7-mers of the homodimer and above). In some embodiments, the protein composition ses no more than 35% of (Tiler—2.045 present as the highest order multimers. in some embodiments, the protein composition comprises at least about 35% of GLu —45 present as tetrarners, ers, hexamers, and 7—mers (£18., at least 35% of the total GL- 2045 ition is comprised of fractions 443, ). in some embodiments, at least about 35% of GL—ZO45 is present as trimers of the homodiiner and above (3.8., at least 35% of the total (EL-2.045 composition is comprised of fraction 3 and above). in some embodiments, at least about 35% of GL—ZO45 is present as trimers, ers, pentamers, or hexamers of the homodimer (216., at least % of the total (lL—2045 composition is comprised of fraction 3—6). in some embodiments, at least about 35% of (EL—2045 is present as tetraniers of the homodimer and above tie, at least 35% [Annotation] yzus [Annotation] yzus of the total GLu2045 composition is comprised of fraction 4 and above). in some embodiments, at least about 35% of GL~2045 is present as tetrarners and pentamers of the honiodimer (ale, at least % of the total (lL—2045 composition is comprised of ons 4 and 5). In some embodiments, at least about 35% of GLuZOA‘lS is present as pentamers of the homodimer and above (116., at least % of the total Gig—2045 composition is comprised of on 5 and above). in some embodiments, at least about 35% of GL—Ztl45 is present as pentarners and hexarners of the mer (he, at least 35% of the total GL~2045 composition is comprised of fraction 5 and 6).

In some embodiments, at least about 35% of GL~2045 is present as hexamers of the hoinodinier and above (216., at least 35% of the total GL~2045 composition is comprised of fraction 6 and above). In some embodiments, at least about 35% of 45 is present as 7wmers of the homodimer and above (lie, at least 35% of the total GL—2045 composition is comprised of fraction ‘7 and above). For example, the optimized manufacturing methods described herein may result in a (EL-2045 protein composition wherein 40%, 45/9, 50%, 5.5%, or greater of the (EL—2045 is present as pentamers of the homodimer and above. Current manufacturing methods for Fc~ containing eutics (cg, monoclonal antibodies) have focused on increased protein titer and yield through the downstream filtration steps. These methods do not generally consider the effects of the cturing process on the multimerization of the chcontaining protein and, in stark contrast to the methods described herein, seek to minimize protein aggregation and niultimerization. Surprisingly, culture ions tl’iat result in the highest protein yields of GI..- 2045 do not necessarily result in the higl‘iest percentage ot‘ Gl_.-2045 present as multimers. As such, the data described herein demonstrates that manufacturing variables that affect total protein titer are, at least in part, independent from variables ing ierization profiles. Therefore, a person of skill in the art would not be able to predict which upstream manufacturing conditions would affect (EL—2045 niultimerization based on the t state of the art. {8084} For example, established protocols for recombinant n production with Chinese hamster ovary (Cl-10) cells e for a temperature shift from 37") C to 3lO C on a specific day of culture (Ouguchi er oi, Cytotechnology, 52(3), pp. 199—207, (2006); Masterson and Smales, Pharmaceutical cessing, 2(l), pp. 49~6l, ). l-lowever, the present inventors found that, in contrast to what was described in ()uguchi, er £21., a temperature shift from 37" C to 32. 5° C resulted in nance of cell density and high cell viability while optimizing for protein titer. r, the inventors found that shifting the temperature based on cell density, rather than [Annotation] yzus [Annotation] yzus on a given day of culture as previously described, resulted in enhanced (EL-2045 protein titers of nearly 10 g/L {8985} Furthermore, the present inventors have discovered that nance of the optimized inerization profile resulting from optimized upstream manufacturing s relies in part on optimized downstream manufacturing methods (6.5;, affinity chromatography and/or ion~exchange chromatography). Monoclonal antibody (mAb) and Fe fusion protein filtration and purification techniques are extensively described and commonly used, However, when applied. to GL~2045, these techniques result in ictable modifications of the GL~2045 multimerization profile. For example, the present inventors have surprisingly discovered that most protein A columns are not suitable for purifying 45, despite their routine use in purifying mAh and Fe fusion proteins, as demonstrated. in e 8. protein A is an ely expensive reagent, costing millions of dollars for use in ance with good manufacturing practices (Glut?) purification of a single drug, and needing to be re—used as many as Hit) or more times in order to be economically . Like mAhs and PC fusion ns, 45 binds protein A, however, unlike inAhs and Fe fusion proteins, Gl_,—2045 does not completely dissociate from protein A with normal elution steps due to the avid binding of Gin—2645 to protein A. The present ors have ctedly discovered that utilization of protein A columns for the purification of GL~ZG45, wherein. the optimal multimerization profile is maintained, requires a more frequent column cleaning schedule, {0086} The present inventors have further discovered that protein A column Clean—in—Place (Cl?) procedures commonly used in the art unexpectedly result in a change in the Gin—204:3 in ultinierizati on profile. Normal (I'll? procedures entail column ng at the end of a purification run, which may involve us cycles of protein supernatant passing through the column.

However, the present inventors have discovered that the high avidity of (ELEMS results in a laclr of 45 dissociation from protein A, Consequently, the binding sites of protein A remain occupied, preventing GLuZGL‘lS binding in subsequent cycles and resulting in a loss of the lioinodimer, Therefore, in contrast to ols utilized with inAlis or Fc fusion protein, the present inventors have unexpectedly discovered that with GL_2045, (Ill? cleaning of Protein A columns must he done more frequently than is done with a monoclonal antibody or he fusion protein and with a highly stringent regeneration buffer, such as 0.5 M Natfil. ation] yzus [Annotation] yzus {8987} pl—l elution gradients are commonly used with protein A columns to optimize protein yield during purification. The present ors have surprisingly discovered that pll elution gradients used in the art to optimize protein A column yields of monoclonal antibodies or Fc fusion proteins can cause an undesired loss ofhomodimer or higher order multimer components of GLUZOLlS, thus ng the multimerization profile of GL~2045 (demonstrated in e 10).

As such, the t inventors have also determined a means of using the elution nt technique to optimize the combination of yield and multimerization of 45. Additionally, the t inventors have surprisingly discovered that pH elution gradient can he applied to protein A column for a novel purpose namely to separate the largest, highly ordered GL~2045 multimers from homodimer aggregates (as demonstrated in Example ll). {9088} The present inventors have also discovered that when using ion exchange columns commonly used in the art to purify onal antibodies and Fe fusion proteins, such as anion exchange and cation exchange, s in pH and/or salt can change the multinierization profile of Gl_,—2045, as demonstrated in Example l2. This stands in stark contrast to a monoclonal antibody or Fe fusion protein where changes in salt or pH may result in the loss of a small amount of protein but no change to the composition. of the drug. Additionally, the present inventors have singly discovered that adjustments of salt and/or pH can he used for a novel purpose, to separate the largest, highly ordered (EL-2045 multimers from aggregates of homodimer that may he of similar molecular mass. As trated in Example l3, the inventors use a functional assay to prove that removal of disordered aggregates from the higl‘iest order multimer fractions is associated with higher potency and a more highly purified GL-leélS product, {9089} l-l'ydrophohic interaction (BIC) columns are commonly used in the art to purify monoclonal dies and Fe fusion proteins through a variety of mechanisms including high- yield capture, polishing monoclonal antibodies, removing truncated species from fullulength forms, separating active from inactive forms, and clearing of viruses. lilowever, when used in the context of GLuZOZlS, the inventors have found that standard l—llC columns are unpredictable. As trated in Example l4, 7 l-llC columns comprised of different matrices are associated with widely differing e rates, ranging from loll/E: to 62%, despite the same supernatant and the same buffer being used with all columns. {@996} Further, the present inventors predict that changes in buffer can change the inultirnerization profile of Gin—“045. This stands in stark st to a monoclonal antibody or Fe [Annotation] yzus [Annotation] yzus fusion protein where changes in buffer would result in the loss of a small amount of protein or less perfect polishing but no change to the fundamental composition of the drug. {8991} Furthermore, the present ors have discovered that because the GL—ZOL’lS homodimer is comprised of lgGl Fc and a multimerizing domain that causes highly ordered niultiiners to be formed, GLwZOItS binds avidly to all or nearly all the many ligands and targets that bind without avidity to a native lgGl Fe mer. This includes, not surprisingly, all the low affinity Fc receptors and complement Clq as well as protein A and protein G used commonly in purification columns. r, this avid binding also results in less desirable potential target binding, for example, endotoxin. For this reason, the present inventors have determined that a multiple step purification process is desirable, including purification of GL~2045 by protein A, and polishing of protein A—purified 5 by at least one or more of cation exchange chromatography, anion exchange flow through, and hydrophobic interaction columns. in a preferred embodiment, a four—step purification process is desirable, ing purification of GL— 2045 by protein A, and polishing of protein A—purified Glam/$5 by all three of cation exchange tography in binding niode, anion exchange in flow h mode, and hydrophobic interaction s in either binding or tlow through mode This four-step purification. process is outlined in Example 15. One of skill in the art will readily understand that additional filtration steps, including depth filtration. and iltration steps may be added at any point, before, , or after the process bed in Example l 5 to further purify the Gil—2045 composition.

Upstream icturmg {9992} Generally speaking, upstream manufacturing methods are methods in which biological. materials are ated and grown in culture, under controlled conditions, to manufacture certain types of protein biological ts (eg. Gil—2045) As used herein “upstream manufacturing methods” specifically refers to methods for recombinant production of a protein without nce to subsequent purification and filtration steps that are generally categorized as downstream manufacturing methods. Upstream cturing methods with alterations or changes aimed at optimization of a specific protein characteristic (cg. multimerization efficiency) are referred to herein as “optimized upstream manufacturing methods,” l aspects of upstream protein manufacturing may be optimized (eg, changed or altered to achieve a desired ) to result in a final protein product with specific characteristics. Aspects of upstream ation] yzus [Annotation] yzus recombinant protein production that may he optimized can e, but are not limited to, composition of the expression vector ng the protein, cell type, basal media, media additives including feeds, feed schedule, passage schedule, culture temperature, temperature shift, humidity, degree of aeration, pl-l, seeding cell density, CO2 level, and/or oxygen level. in some embodiments, the optimized upstream manufacturing s described herein result in the tion of a high titer of GL~2045 with an increased percentage of higher order multimers compared to GIL—2045 ed by nonnoptimized upstream cturing methods. {9093] in some aspects of the ion, Chinese hamster ovary (CHO) cells are transfected with an expression vector encoding {EL—2045. In some aspects, ion of the GL— 2045 expression cassette into the genome is mediated by the piggyBac transposon, n 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 a piggy‘flac transposase mediates gene integration into regions of the genome that are actively transcribed, resulting in the generation of cell lines with stable and enhanced gene expression compared to standard ection methods (See US 2010/03 l l l 16 and Matasci er of, Biotechnol. Bioeng. V. ltig, pp 214lw2140, (ZOl l), herein incorporated. by reference). The piggyback system normally increases protein production due, at least in part, to a. high number of integrated transgenes. However, the present inventors selected a high titer, high viahility clonal cell line wherein the transgene insertion rate was relatively low (tag, approximately ll copies determined by UV spectrophotometry); however one of skill in the art would understand that the use of selective antibiotic pressure also allows for the use of ene insertion rates of greater than about 50 inserted copies or more than about lOO inserted copies. In some s, the expression vector comprises a nucleic acid encoding a leader peptide tag. SEQ ll) NO: l). in some aspects, the expression vector further ses an antibiotic resistance gene to allow for the selection of successfully ti'ansfected CHO cells. In some aspect of the invention, successfully transfected CHO cells are generated in the ahsence of antibiotic selection (See US 20l0/03llllol. in some aspects, the sion vector further comprises a transcriptional promoter to promote high level expression of GL~2045 (cg. a Clle promoter). {0094} in some embodiments, CHO cells transfected with a GLuZOI-lfi expression vector are cultured in a ctor. ln some embodiments, the CEO cell line, of which there are many variants, is carefully selected so that once stably transfected with a (EL—2045 expression vector [Annotation] yzus ation] yzus using techniques that cause insertion entially at transcriptionally active sites. In some embodiments, the culture conditions applied to said transfected select (ll—IO cell line allows for the culture protocol to continue longer than by rd cturing methods without adversely affecting cell viability. Standard manufacturing methods can average l2 days in a bioreaetor, at which point there is a se in cell viability due to an increase in cellular debris present in the culture. In addition to being associated with a loss of cell viability, cellular debris dramatically increases challenges associated with filtration and purification. In some embodiments of the present ion, cells are seeded in a bioreactor at a predetermined cell y and cultured for > l2 days. In some embodiments, the cells are ed in the bioreactor with acceptable viable cell density for l3, 14, l5, l6, l7, IS, 19, 20,, ill, or more days. {9095} In some embodiments, ActiCIIO l’ is used as the basal media for optimized am manufacturing of optimally manufactured (3“,I.,-20/-'l5~ The terms “ActiCHO l?” and “optimized media,” as used interchangeably herein, refer to the. commercially available ActiCHO® base media. (“ActiCHO F,” GE Healtlicare), any substantial copies thereof, or media that comprises substantially the same. constituents in substantially the same quantities as ActiCIIO P. ActiCHO P has also recently been marketed by GE as Hyclone “AetiPro,” a nearly identical product to ActiCHQ P and which is an equivalent reagent for the purposes of these sures In some ments, ActiCHO® Feed A and Feed B (also recently marketed by GF as Hyclone “Cell Boost, 7a” and I-chlone “Cell Boost 7b,” which are identical products to ActiClI0® Feed A and Feed B and are equivalent reagents for the purposes of this disclosure) are used in addition to the basal media. The terms “ActiCI-IO Feed ”t” or “optimized feed ”I,” as used interchangeably herein and “ActiCl-IO Feed B” or “optimized feed B”, as used interchangeably herein refer to the commercially available IO® Feeds (GE l-Iealthcare), substantial copies thereof, or feeds that se substantially the same constituents in substantially the same quantities as ActiCl—IQ Feed A and/or ActiCl—IO Feed B. In some aspects of the invention, CHO cells 'transfected with a GL- 2045 sion vector are fed with ActiCHO Feed A and Feed B every day. In some aspects of the invention, (El-IO cells transfected with a (EL-2045 expression vector are fed with ActiCl-IO Feed A and Feed B every other day. In some aspects of the invention, {II-IO cells transfected with a (EL-2045 expression vector are fed with ActiC‘lrlO Feed A and Feed B via continuous feed. {8996} In some embodiments of optimized upstream manufacturing methods, (II-IO cells transfected with a GEL-2045 expression vector are grown to a specific cell y prior to shifting [Annotation] yzus [Annotation] yzus the temperature. In some aspects, the CHO cells are grown to a density of about 5 30 million cells/m1; prior to shitting the temperature. 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 shifting the temperature. in some aspects, CHO cells are grown to a density of about 10 25 million eells/mL prior to ng the temperature. In some aspects of optimized manufacturing methods, CHO cells are grown to a density of about it) — 15 million cells/mL prior to shifting the temperature. 313097} In some embodiments, CHO cells transfected with a GLnIZOélS expression vector are cultured at 37° C :: 1°C until reaching a ermined cell density. in some aspects, the ature is shifted to 32.50 C it 1°C after the cells reach a predetermined cell density. This aspect is in contrast to previously described culture methods for recombinant protein production, wherein cells are cultured at 37° C for a predetermined number of days, after which the temperature is often shifted to 3l°C (Ouguchi at al, Cytotechnology, 52(3), pp. 199—207, (2006); son and Sinales, Pharmaceutical Bioprocessing, 2(1), pp. 49—61, (2014)). The present inventors have determined that shifting the temperature from 37° C 21?: 1°C to 32.5" C at: 1°C based on cell density, rather than culture time, unexpectedly provides the combination of increased ity, improved cell density, and a substantial increase in protein titer relative to standard upstream manufacturing methods. In some embodiments, (fl-IO cells transfected with a (EL—2.04:3 expression vector are subiected to a. double ten'iperature shift. in one ment, transfected CHO cells are cultured at 37° C :1:- l°C and shifted to 34° (I :1: 1° C before reaching peak viable cell density. in a red embodiment, this temperature shift occurs while the CHO cells remain in log growth phase. in an especially preferred embodiment, the initial temperature shift occurs on day 3 or 4 of culture. In another embodiment, transtected (El-l0 cells are cultured at 37° C :5; l°C until reaching a predetermined cell y, at which time the temperature is shifted to 34° C j: 1° C. in a red ment, the cell density is between 5 — 20 million cells ml at the first temperature shift. in an especially preferred embodiment, the cell density is between 8 _ l5 million cells / ml at the first ature shift. in some embodiments, the second ature shift occurs at day '7 :l; 1 day. in some ments, the temperature is then further shifted to 31°C. In some embodiments, this second temperature shift is performed at about day 4 post initial temperature shift.

Downstream i’l/[amrfilclurmg [Methods [Annotation] yzus [Annotation] yzus {8993} In some embodiments, harvesting of {iii—2045 is accomplished by downstream manufacturing methods. In some embodiments, downstream manufacturing s are employed in combination with the optimized upstream manufacturing methods described herein to remove or isolate a specific protein fraction (cg, removal of unordered high molecular weight aggregates of the mer 45). As used herein, “downstream manufacturing methods” are protein purification and filtration steps med on protein supernatants to generate a protein composition of a desired purity and/or concentration. In some embodiments, downstream manufacturing methods have been optimized for the purification and filtration of (EL-"2045 to result in and/or maintain a particular inultimerization profile of GLEN, referred to herein as “optimized downstream manufacturing methods.” {9099} In some embodiments, GL—ZO45 is purified by affinity chromatography. In some embodiments, GLQOZIS is purified using protein A, columns. As described above, protein A, columns are very expensive and are reused a. considerable number of times in order to become economically . The re—use of protein A columns necessitates “regenerating” the protein A, column in order to maintain protein binding capacity. As used herein “regenerating” or “regenerate” refers to the removal of bound n from. the protein A column that was not removed during the elution process. In some embodiments, the protein A, column must be regenerated more often during GI...—20-45 purification than ted in the manufacturer instructions or more often than is normal in the art for ing onal antibodies or Fc fusion proteins. In some embodiments, the protein A column must be regenerated at least twice as often as recommended by the manufacturer. In further embodiments, the protein A column must be regenerated in between each sive round of g of (EL—2045 supernatant over the column In such embodiments, purification of 45 with a n A affinity column necessitates the use of a high stringent regeneration buffer to remove avidly bound (EL—2045 multimers from the n A column and regenerate the full binding capacity of the protein A column. In preferred embodiments, the high stringent ration buffer does not cause degradation of the protein A column or is associated with little degradation of the column. In some embodiments, the highly stringent regeneration buffer comprises a soluble base. In some embodiments, the base is sodium hydroxide (NaOH), In some embodiments, the regeneration buffer has an NaOI-I concentration of greater than 0.3 M NaGI-l. For example, the high stringent ration buffer may be greater than ation] yzus [Annotation] yzus 0.35 Ni, 0.4 M, 0.5 M, 0.6 M, 0.7 Ni, 0.8 M, 0.9 M, ll) M, or more NaOH. in ular embodiments, the concentration of NaGl—l in the ration buffer is 0.5 M Nagl-l, } in some embodiments, elution of (EL-2045 from a protein A affinity column is optimized to remove or reduce the amount of high lar weight, unordered aggregates ofGLm 2045 from the drug substance. In some embodiments, GLUZO45 is eluted from a protein A column by an elution gradient (cg, a pH elution gradient). 3130191} In some embodiments, optimized downstream cturing methods for Glyn 2045 se a multiple step purification process comprising purification by affinity chromatography (6.g, protein A affinity chromatography) and at least one or more polishing steps selected from cation exchange tography, anion exchange chromatography, and hydrophobic ction columns. ln a preferred. ment, a four step cation process for (EL-£045 is used d of the two or three step purification process commonly practiced in the art, comprising purification by affinity chromatography (cg, protein A ty chromatography) polishing by each of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction columns. The term “polishing” classically refers to post—protein A purification removal of remaining impurities including aggregates, xin, DNA, and/or Viruses. Additionally, with respect to Gil—2045, “polishing” additionally means controlling the percent of homodimer and specific higher order multimers such as through the use of these same chromatographic techniques. {00192} In some embodiments, Gig-2045 is polished by ion exchange chromatography (tag. cation or anion exchange). in some embodiments, polishing of (EL—2045 by ion exchange chromatography is med with an elution buffer that reduces and/or minimizes the amount of unordered, high molecular weight aggregates of the Gil-2045 l'ioinodimer that are retained during the postuprotein A purification process. in some embodiments, step elu'tion is performed to elute GL-ZO45 from the protein A column. in some embodiments, a gradient elution is performed to elute {EL—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 may be at least 30, 35, 40, 45, 5t}, 55, 6t}, 75, 100 mM, or more of sodium acetate, In some embodiments, the elution buffer is SOmh/l sodium acetate. in some embodiments, the elution buffer is 50 mM sodium e with the addition of varying amounts of an additional salt buffer (3.g an NaCl buffer). In [Annotation] yzus [Annotation] yzus some embodiments, the additional buffer is a l M > pI-l 5 buffer (“buffer h”). In some ments, the elution buffer comprises at least 30% buffer B. In some embodiments, the n buffer comprises between 30% and 40% buffer B. In some embodiments, the el ution buffer comprises between 35% and 409/15 buffer B. in still further embodiments, the elution buffer comprises n 37% and 3¢% buffer B. in some embodiments, the elution buffer is 3 % +/— 0.5% buffer b. 3130193} In some ments, GLn2045 is polished, using hydrophobic interaction chromatography (BIC). in some embodiments, the HIC column is selected to remove the high molecular weight, unordered aggregates of (EL—2045 (cg, an Octyl EF HIC ). Either flow through mode or binding mode may be performed to purify Gig—2945 with the HIC column. The skilled. artisan understands that adjusting variables such as pH and salt ions will determine whether GL—2045 binds to the HIC resin or tlows through the column. in some embodiments, the HIC column is selected to purify a specific fraction of 45, such as the homodinier and/or the higher order multimers (rag, a Butyl HP and/or a Phenyl HP column). In some embodiments, it may be desirable to isolate a. specific fraction of (EL-2045 for the treatment of a particular disease indication For example, HIC columns may be used to generate drug substances that are substantially comprised of a specific GI...—2045 fraction (cg, a drug substance that is predominantly comprised of 61.,»2045 homodimers, a drug substance that is predominantly comprised of dimers of the homodiniers, a drug substance that is predominantly con'iprised of higher-ordered multimer of (EL—2045, etc), Separation of {ill-2045 fractions into separate products may be ageous for certain disease indications. For example, the (]l_,~2045 imer binds to Fcy'Rl', but does substantially bind to other Felts. As such, the GLuZOL'lS mer may be especially useful in the treatment of es mediated, at least in part, by Fcle signaling, such as peritonitis (Heller at at, I. lmmunol, V. loz, l992) or acute lung injury (Xie et al. J. Immunol, V 188, 2012). Similarly, the trimer of GLnZGa’lS, and ially the dimer and tetramer, may be particularly useful for treating mune diseases (See, WO 20l5/168643} As Clo is hexameric, the pentamer, hexamer, and heptamers may be especially useful in the treatment of complementwmediated diseases. A skilled n will recognize that these are only some of the ways that (Si—2945 fractions may be advantageous for treating certain diseases. {(39184} In some embodiments, the optimized downstream manufacturing methods described herein may be a combination of individual purification and/or filtration techniques. For [Annotation] yzus [Annotation] yzus example, in some embodiments, the optimized downstream methods may comprise purification of GL~2045 by affinity chromatography leg, purification by optimized methods for protein A columns) followed by additional polishing with ion exchange chromatography methods (eg. polishing by optimized cation exchange methods) and/or hydrophobic interaction columns. in some embodiments, the optimized downstream methods described herein comprise a four~step purification process including purification by n A, cation exchange, anion exchange flow through, and hydrophobic interaction columns. ln some embodiments, additional depth filtration and/or iltration steps may also be used. {@9195} Thus, the terms “optimal manufacturing methods” or “optimized manufacturing methods” used interchangeably herein, may refer to optimized upstream and/or optimized downstream manufacturing methods. In some embodiments, the zed manufacturing methods comprise both optimized upstream and downstream s. As such, the terms “optimally ctured stradomer” or ally manufactured Gill-2945.,” as used herein, refer to high titer, high-order multimer dominant Gin—2045, compositions made in accordance with optimized upstream manufacturing conditions and/or optimized downstream manufacturing methods. While the GIL—2045 composition described herein. may he lly produced GL~2045 (219., Gil—204:3 made by the methods described herein), one of slcill in the art will understand that 81.,»2045 compositions that fall Within. the defined multimer patterns described. herein may he achieved by other means. Therefore, a “Gl..»2045 composition” or “recombinant 045 composition” or “purified (EL—2045 composition” refers to a composition comprising (31-4045, including a GL—2045 drug substance, r the composition was made Via optimal manufacturing methods or not. , the terms mer pattern” or “handling pattern” or any like term are used interchangeably and refer to the pattern of rnultimers observed in an analytical assay ofa Gl_,-2045 composition. An exemplary ner pattern is shown in FIG 33 a {801%} In some embodiments, a recombinantly produced GI...—2045 ition with a defined multimer pattern is provided herein. As used , the terms “defined rnultimer pattern” or “defined multimerization pattern” or “defined banding pattern” refer to a pattern of (EL—2045 multimerization that is reproducible and can he described in terms of the percentage of the total (EL-2045 composition present as homodimers, higher order ers, and/or highest order multimers Gne of ry skill in the art will understand that the absolute value of the homodimer and/or er tages may vary based on the analytical method used. By way of example, [Annotation] yzus [Annotation] yzus digital software analysis of an SD8~PAGE gel will yield somewhat different multimer percentages compared with analytical SECul-lPLC of the identical composition. Unless otherwise ied herein, the percentages of hornodirners and ers of the {EL—2045 compositions described herein are expressed as percentages measured by analytical SECd—lPLC methods. in some embodiments, a recombinantly produced GL~2045 is provided in which at least 80% of 45 is present as modirners or “multiniers” (age, dimers of homodimers, trimers of homodimers, etc). In some embodiments, greater than 80% of GL~ZO45 is present as niultiiners. For example, optimized manufacturing s, such as those described herein may result in a (EL—2945 protein composition wherein 80%, 85%, 90%, 95%, or greater of the G ' —2045 is t as multimers. In some embodiments, at least 30% of GLnZtlZlS is present as “highest order multimers,” defined herein as the 7—mer of the homodinier and above. In some ments, no more than 40% of the reconibinantly ed lS is t as highest ordered multimers. One of ordinary skill in the art will also understand that when we talk about “hands,” or “fractions” unless specified otherwise, the number of the band es the number of homodimers present in the fraction.

Thus, for example, band 2 comprises the dimer of the homodimer while band 3 comprises the trimer of the homodimer Thus, for example, band 2 comprises the dimer of the homodimer, band 3 comprises the trimer of the homodimer, band 4 comprises the er of the homodimer, etc.

} As used herein, the term “higher order multimers” refers to the trimers of the homodimer and above (ten, multimers t in fraction 3 and above). As used herein, the term “highest order multimers” refers to the multimers in fraction 7 and above, or fractions including the 7—mer of the homodinier and above. {90198} In some embodiments, a recombinantly produced GL—ZO45 composition is provided wherein the homodimeric traction comprises less than about 20% of the total composition. In some embodiments, the homodimeric fraction comprises about l 2% to about l9%, about 14% to about 19%, about 155% to about l7.5%, or about 14% to about 185% of the total protein composition. In some embodiments, the homodirneric traction comprises about l5,9% or about 16.2 % of the total protein composition. {4919199} In some embodiments, a recombinantly produced Git—2045 composition is provided wherein the dimer of the homodimer fraction comprises about 7/2: to about 13%, about 7% to about 12. 5%, about 7% to about l2%, about 9% to about l l%, or about 9. l % to about l l 7% ation] yzus [Annotation] yzus ot‘ the totai composition. in some embodiments, the dimer of the homodimei' fraction ses about 10% or about ) of the total n composition. {11111111} in some embodiments, a recombinantiy produced GL—ZOZ’iS composition is provided wherein the trimer of the homodimer fraction comprises about 5.5% to about 11%, about .5% to about 10%, or about 6.5% to about 8% of the totai composition. In some embodiments, the trimer of the homodimer fraction ses about 7% or about 7% of the totai protein composition. {110111} 1n some embodiments, a recombinantiy produced GAL-2045 composition is ed wherein the tetranier of the hoinodimer fraction comprises about 10% to about 16%, about 1 % to about 16%, about 13% to about 15%, or about 12.4% to about 15.1% of the total composition, in some embodiments, the tetranier of the homodimer on comprises about 14% or about 14.3% of the totai protein composition {1311112} In some embodiments, a. recombinantiy produced 1314—2045 composition is provided wherein the pentamer of the hornodimer fraction comprises about 6% to about 9%, about 7% to about 8%, or about 7.1% to about 8.2% of the totai composition. in some embodiments, the dimer of the pentamer fraction comprises about 7% or about 7.5% of the totai protein composition. {1111113} in some embodiments, a. recombinahtiy ed 1314—2045 composition is provided wherein the er of the homodimer fraction comprises about 1.0% to about 14%, about 12% to about 13%, or about 12.1% to about 13.2% of the total COHIpOSiEiOfl. in some embodiments, the hexamer of the homodimer fraction comprises about 12.7% or about 1.26% of the totai n composition. {1111114} In some embodiments, a recombinantiy produced (TEL-2045 composition is provided wherein the highest order muitimer fraction comprises at ieast about 28% of the total composition. In some embodiments, the highest order muitimer fraction comprises no more than about 35% of the total protein composition. In some embodiments, the highest order muitimer fraction ses about 30% to about 34%, or about 28.6% to about 35.1% of the total protein composition. in some embodiments, the highest order muitimer fraction ses about 31.41%) or about 31.9% of the totai protein composition. 1n some embodiments, a recombinantiy produced {EFL—2045 composition is provided n the homodimeric fraction comprises less than about % of the totai composition; the highest order multimer fractions comprise at ieast about 289/13 of the total composition; the dimer of the homodimer fraction comprises from about 7% to about 13% [Annotation] yzus [Annotation] yzus ot‘ the totai composition; the trimer of the homodirner fraction comprises from about 5% to about 11% of the total ition; the tetramer of the homodimer fraction comprises from about 30% to about 16% of the total composition; the pentamer of the homodimer fraction comprises from about 6% to about 10% of the total composition; the hexanier of the homodimer fraction ses from about 10% to about 14% of the total on; the dimer of the homodimer through hexamer of the homodimer fraction comprises from about 40% to about 60% of the total composition; the trimer of the mer through the hexamer of the homodimer fractions comprise from about 32% to about 50% of the totai composition; the er of the mer through the hexamer of the homodimer fraction se from about 30% to about 37% of the total composition; the pentamer of the homodimer through the hexainer of the homodimer fraction comprise from about 18% to about 23% of the total composition; or any combination of the forgoing. {(39115} In some embodiments, a inantiy ed GL~2045 composition is provided, wherein the approximately 80% of the totai GL~2045 composition comprises the dimer of the homodimer and above (116., band 2 and above). In some embodiments, approximateiy 60 80%, 62—30%, or 60—78% of the total inantiy—produeed (EL—2045 composition comprises the trimer of the homodimer and above (1265., bands 3 and above). In some embodiments, about 54- 7694), about 54'72%, about 56-7 %, or about 54-67% the total recombinantiy produced GI...—2045 composition comprises the tetramer and above (Le, bands 4 and . In some embodiments, a GL-ZO45 composition is provided, wherein approximateiy 44—60%, 44—57%, or 44~5i% of the total composition comprises the pentamer and above (5.8., bands 5 and above). In some embodiments, a (31,—2045 composition is provided, wherein approximately 38-51% of the total composition comprises the hexamer and above (519., bands 6 and above). {@6116} in some embodiments, a recombinantly ed Giff/2.045 is ed wl'ierein bands 2%) of the composition (file, the dimer of the homodimer through the er of the homodimer) comprise about 39—61% or about 44—60% of the composition. in some embodiments, a recombinantiy produced (EL—2045 is provided, wherein bands 3—6 of the composition (113., the trimer of the homodimer through the hexamer of the homodimer} comprises about 32-50% or about 35~48':?/é3 of the composition. In some embodiments, a recombinantiy produced GLu2045 is provided wherein bands 4~6 of the composition (212., the tetramer of the homodimer through the hexainer ot‘the homodiiner) comprises about 26—3 ‘% or about 30—39% of the composition. in some embodiments, a recombinantiy produced (EL—2045 is provided wherein bands 5—6 of the [Annotation] yzus [Annotation] yzus composition (tie, the er of the homodimer through the hexamer of the hornodirner) comprises about 152394; or about 18—23% of the composition. {@9117} t being bound by theory, the least active components of lfi in binding to low ty Fc receptors and to Clq are the homodimer and the dimer of the homodimer. A skilled, artisan will y appreciate that one can use the optimized chromatographic methods described herein, or similar purification techniques, to reduce the amount of homodimer or homodirner and dimer in the final product. The skilled artisan will thus know that doing so will alter the percentages of the multimers disclosed herein. By way of example and without limiting the generality of the foregoing, if the skilled, artisan were to remove 50% of the honiodimer in the purification s, or homodimer and the dimer, then the percentage of each remaining multimer (rte, trimers, tetramers, pentamers, hexamers, 7—niers, etc.) would. correspondingly se.

Removing 90% of the homodimer and 50% of the dimer will decrease the total protein present in the final product by approximately 20% +/—5%, and will therefore increase the percentages of the trimer, tetranrer, pentamer, bexamer, and 7—mer represented as a percent ot‘tlie total protein. {(39118} What is more, one of skilled in the art will further recognize that current tography techniques do not generally permit l or reduction of a single multinier hand, such as the highest order multimers, without simultaneously removing, to some degree, the adjacent bands, such as the hexamer and to a lesser extent the pentamer. ore, the skilled artisan will know that the observed compensatory increase in the percentage of any given multirn er or honiodiiner as a result of removal or reduction of the highest order multimers will increase by a greater degree the farther the given rnultirner is from the fraction of (file—204:3 that is d (cg, the tage of the homodimer will se by a greater degree than the increased percentage observed for the hexaniers when the t order multimers are removed or reduced). ln any case, the cumulative increase in multimer percentages of the remaining multimers should equal the multimer percent for the removed fractions, subject to some variability attributable to analytical method.

Pharmaceutical Compositions {08119} 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, hue-cal, rectal, l, ophthalmic, subcutaneous, intramuscular, intraperitoneal, [Annotation] yzus [Annotation] yzus intravenous, intraarterial, intratumoral, spinal, intratliecal, intra—articular, intra—arterial, sub— arachnoid, sublingual, oral mucosal, bronchial, lymphatic, intrauterine, subcutaneous, intratumor, integrated on an implantable device such as a suture or in an table device such as an table polymer, intradural, intracortieal, or dermal. Such compositions would normally be administered as pharmaceutically able itions as described herein. In a preferred embodiment, the ed, optimally manufactured, stradomer is administered intravenously or subcutaneously. {9012-9} The term "pharmaceutically acceptable carrier" as used herein includes any and all solvents, dispersion media, gs, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the vectors or cells of the present invention, its use in therapeutic itions is contemplated. Supplementary active ingredients also can be incorporated into the compositions {(39121} The Clo—2045 compositions of the present invention may be formulated in a l or salt form. Pharinaceutipally—acceptable salts include the acid addition salts d with the free amino groups of the protein) which. are formed with inorganic acids such as, for example, hydrochloric or oric acids, or such organic acids as acetic, oxalic, tartaric, mantlelic, and the like. Salts formed with the free carboxyl groups can also be derived from inorganic hases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as pylamine, hylamine, histidine, procaine, and the like. {(38122} Sterile injectable solutions are prepared by incorporating the optimally manufactured (314—2045 in the required amount in the appropriate solvent with various of the other ients enumerated above, as required, followed by filtered sterilization. in some embodiments, the sterile injectable solutions are formulated for uscular, subcutaneous, or intravenous administration. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic sion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum—drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile—filtered solution thereof.

[Annotation] yzus [Annotation] yzus } Further, one ment is a (EL—2045 composition suitable for oral administration and is provided in a pharmaceutically acceptable carrier with or without an inert diluent. The carrier should he assimilable or edible and includes liquid, semi—solid (cg, pastes), or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the eutic effectiveness of an optimally ctured mer preparation contained therein, its use in an orally administrable optimally manufactured stradomer composition for use in practicing the methods of the present invention is appropriate. Examples of rs or diluents include fats, oils, water, saline solutions, , liposomes, resins, binders, fillers and the like, or combinations thereof. The term "oral administration" as used herein includes oral, buccal, enteral or astric administration. {90124} In one embodiment, the 45 composition is combined with the carrier in any convenient and practical manner, ie., by solution, suspension, emulsification, admixture, encapsulation, microencapsulation, absorption and the like. Such procedures are routine for those skilled in the art. {£39125} in a specific embodiment, the GL~ZO45 composition in powder form is combined or mixed thoroughly with a semi-solid or solid carrier. The mixing can he carried out in. any convenient manner such as grinding. Stabilizing agents can he also added in the mixing process in order to protect the composition from loss of therapeutic actiVity (cg, through denaturation in the stomach). Examples of stabilizers for use in an orally adniinistrable composition include s, antagonists to the secretion of stomach acids, amino acids such as glycine and lysine, carbohydrates such as dextrose, rnannose, galactose, fructose, lactose, sucrose, e, sorhitol, rnannitol, etc, lytic enzyme inhibitors, and the like. More preferably, for an orally administered composition, the stabilizer can also include antagonists to the secretion of stomach acids. {(130126} Further, the (EL—2045 composition for oral stration which is ed with a semisolid or solid carrier can be further formulated into hard or soft shell gelatin capsules, tablets, or pills. More preferably, gelatin capsules, tablets, or pills are enterically coated. Enteric coatings prevent ration of the composition in the stomach or upper bowel where the pl-i is acidic. See, US. Pat. No. 001, Upon reaching the small intestines, the basic pH therein dissolves the coating and permits the composition to be released to interact with intestinal cells, ag Peyer‘s patch M cells. ation] yzus [Annotation] yzus {89127} in another embodiment, the (EL—2045 composition in powder form is combined or mixed thoroughly with materials that create a nanoparticle encapsulating the immunologically active biomiinetic or to which the immunologically active biomimetic is attached. Each nanoparticle will have a size of less than or equal to 100 microns. The nanoparticle may have mucoadhesive properties that allow for gastrointestinal absorption of an immunologically active etic that would otherwise not be orally bioavailahle. {80128} In another embodiment, a powdered composition is combined with a liquid carrier such as water or a saline solution, with or without a stabilizing agent. {@9129} A specific (EL—2645 formulation that may be used is a solution of immunologically active biomimetic protein in a nic ate based buffer that is free of potassium where the composition of the buffer is as follows: 6 mM sodium phosphate monohasic monohydrate, 9 mM sodium phosphate dihasic heptahydrate, 50 mM sodium chloride, pH 70 +/— 0.1. The concentration of immunologically active hiomimetic protein in a hypotonic buffer may range from l0 ug/mL to 100 mg/mL This formulation may be administered via any route of administration, for example, but not limited to intravenous administration. {80139} Further, a GL-«2045 composition for topical administration which is combined with a semi—solid carrier can he further formulated into a cream or gel ointment. A preferred carrier for the formation of a gel ointment is a gel polymer. Preferred, polymers that are used to manufacture a gel composition of the t invention include, but are not limited to carbopol, ymethyl— cellulose, and ic polymers. Specifically, a powdered Fc multimer composition is combined with an aqueous gel containing a rization agent such as Carhopol 930 at strengths between 0. 5% and 5% wt/volume for application to the skin for treatment of e on or beneath the skin, The term al administration” as used herein includes application to a dermal, epidermal, aneous, or mucosal surface. {@0131} Further, a. (EL—2045 composition can be formulated into a r for aneous or subdernial implantation. A preferred formulation for the implantable drug—infused polymer is an agent Generally ed as Safe and may e, for example, cross—linked n (Samantha Hart, Master of Science Thesis, “Elution of Antibiotics from a Novel Crosslinhed Dextran Gel: Quantification” Virginia Polytechnic Institute and State University, June 8, 200‘?) dextran—tyramine (Jin, et a]. (ZOlO) Tissue Eng. Part A, l6(3)12429—40), dextran—polyetl'iylene glycol (hikes, er al. (20l 0) Tissue Eng, Part A, l6(2):565-73), or dextran—gluteraldehyde [Annotation] yzus [Annotation] yzus (Brondsted, at all. (1998) J. Controlled Release, 532743). One skilled in the art will know that many similar polymers and hy drogels can be formed incorporating the stradorner fixed within the polymer or gel and lling the pore size to the desired diameter. {@6132} Upon formulation, solutions are administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective to result in an improvement or remediation of the symptoms. The formulations are easily administered in a variety of dosage forms such as ingestible solutions, drug release capsules and the like. Some ion in dosage can occur depending on the condition of the subject being treated. The person responsible for administration can, in any event, determine the appropriate dose for the individual subject.

Moreover, for human administration, preparations meet ity, general safety and purity standards as required by FDA Center for Biologics Evaluation and Research standards. {(39133} The route of stration will vary, naturally, with the location and nature of the disease being treated, and may include, for example intradermal, ernial, subdermal, parenteral, nasal, intravenous, intramuscular, intranasal, subcutaneous, percutaneous, intratracheal, eritoneal, intratumoral, ion, lavage, direct injection, intra—rectal, and oral admini stration. {00134} In one embodiment, the GL—ZOL’ES composition intravenously, subcutaneously, orally, intraperitoneally, sublingually, buccally, transdermally, rectally, by subdermal implant, or intran'iuscularly In particular embodiments, 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 trig/Kg to about l000 trig/Kg. In a further embodiment, the optimally manufactured stradomer is administered at about 0.0l trig/Kg to about l00 Kg, In yet a further embodiment, the optimally ctured stradomer is stered at about 0.l trig/Kg to about 20 mg/Kg In still a further embodiment, the optimally manufactured stradomer is administered at about 1 Eng/Kg to about 10 org/Kg. in still a further embodiment, the optimally manufactured stradomer is administered at about 2 mg/Kg to about 5 trig/Kg. The optimally manufactured stradomer may be administered at least once daily, weekly, biweekly, monthly, or sometimes longer intervals. A biphasic dosage n may be used wherein the first dosage phase ses about 0. l El/Es to about 300% of the second dosage phase. {00135} In a further embodiment, the GL2045 composition is stered before, during or after administration of one or more additional pharmaceutical and/or therapeutic agents. In a [Annotation] yzus [Annotation] yzus further embodiment the additional pharmaceutically active agent comprises a steroid; a biologic antiuautoimmune drug such as a monoclonal dy, a fusion protein, or an antiucytolrine; a non- biologic anti—autoimmune drug; an inununosuppressant; an antibiotic; and anti—viral agent; a cytokine; or an agent otherwise capable of acting as an immunennodulator. In still a further embodiment, the steroid is prednisone, prednisolone, cortisone, dexamethasone, mometasone testosterone, en, oxandrolone, fluticasone, budesonide, beclamethasone, rol, or levalhuterol. In still a further embodiment, the monoclonal antibody is eculizuma‘o, ocrelizumab, infliximala, adalimumah, mah, tocilizuinab, golimumah, umala, LY2127399, helimumab, veltuzuinah, mepolizumab, necitumumab, nivolumab, dinutuximab, secultinumab, evolocuinah, hlinatuinomab, pembrolizumah, ramucirumah, vedolizumah, siltuximab, ohinutuzumah, adotrastuzumab, cnma‘o, pertuzumah, brentuximal), ipilumumab, denosumah, oanakinumah, ustekinumah, catnmaxomab, tanibizumab, panitumumab, zumab, hevacizumab, cetuximah, etalizumah, omalizumab, toitumomah—lli‘i l, alemtuzumab, gemtnzumah, trastuzumah, palivizumah, basilixumah, daclizumab, mah, morononomah, vedotin, ibritumomab tiuxetan, motavizumab, or certolizumah, In still a r ment, the fusion protein is etanercept or abatacept. In still a further ment, the anti-cytokine biologic is anakinra. In still a r embodiment, the anti~rheumatic non—biologic drug is cyclophosphamide, methotrexate, azathioprine, hydroxychloroquine, leflunomide, minocycline, organic gold compounds, fostamatinib, tot‘aoitinih, etoricoxi‘o, or sulfasalaxine. In still a further embodiment, the immunosuppressant is porine At tacrolimus, mus, mycophenolate mofetil, everolimus, ()KT3, antithymocyte globulin, basiliximah, daclizumumah, or alemtuzumah.

In still a further embodiment, the optimally manufactured mer is administered before, during or after administration of a chemotherapeutic agent. In still a further embodiment, the optimally manufactured stradomer and the additional therapeutic agent display therapeutic y when administered together. in one embodiment, the optimally manufactured stradomer is administered prior to the administration of the additional therapeutic against. in another ment, the optimally manufactured stradomer is administered at the same time as the administration of the additional therapeutic agent. In still another embodiment, the optimally manufactured stradomer is administered after the administration with the additional therapeutic agent. {@9136} In one embodiment, the GL—ZOdS composition is administered covalently fixed to an implantable device In one embodiment, the optimally ctured stradomer is fixed to a ation] yzus [Annotation] yzus suture. in another ment, the optimally manufactured stradomer is fixed to a graft or stent.

In another embodiment, the optimally manufactured stradomer is fixed to a heart valve, an orthopedic joint replacement, or implanted electronic lead. In another ment, the optimally manufactured stradomer is fixed to and embedded within an table matrix. In a preferred embodiment, the lly manufactured stradomer is fixed to and ed within an implantable hydrogel. In one embodiment, the hydrogel is comprised of dextran, polyvinyl alcohol, sodium polyacrylate, or acrylate polymers. In a further embodiment, the optimally manufactured stradorner is administered fixed in a hydrogel with pore sizes large enough to allow entry of immune cells to ct with the fixed stradomer and then return to circulation. In a further embodiment, the pore size of the el is 5 to 50 s. In a preferred embodiment, the pore size of the hydrogel is 2’5 — 30 s. 7} In another embodiment, the GL~ZO45 composition is administered to treat humans, non-human primates (eg, monhe is, baboons, and chimpanzees), mice, rats, hovines, horses, cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, hats, hirds leg, chickens, turkeys, and ducks), fish and es with speciesvspecific or chimeric stradomer molecules. In another embodiment, the human is an adult or a child. In still another embodiment, the lly manufactured stradorner is administered to prevent a complentrant—mediated disease.

In a further embodiment, the stradomer is administered to prevent vaccine—associated mune conditions in companion animals and livestock. {90138} The term ”parenteral administration" as used herein includes any form of administration in which the compound is absorbed into the subject without involving absorption via the intestines Exemplary parenteral administrations that are used in the t invention include, but are not limited to subcutaneous, intramuscular, intravenous, intraperitoneal, intratumoral, intraocular, nasal, or intraartieular administration. {(130139} In addition, the {EL—2045 composition of the current invention may optionally be administered before, during, or after another pharmaceutical agent. {(30146} Below are specific examples of various pharmaceutical formulation categories and preferred routes of administration, as ted, for specific exemplary diseases: {08141} Buccal or sub~llngual dissolvahle tablet: angina, teritis nodosa, {89142} enous, intramuscular, or subcutaneous: myasthenia gravis, hernolytic uremic syndrome (HUS), atypical hemolytic uremic syndrome (aHUS), paroxysmal nocturnal [Annotation] yzus [Annotation] yzus hemoglobinuria (PNl-l), membranous nephropathy, neuromyelitis optica, antibody—mediated rejection of afts, lupus nephritis, membranoproliferatiye glomerulonephritis (MPGN), idiopathic thrombocytopenic pui‘pura, inclusion body myositis, paraproteinemic lgM inating polyiieuropathy, necrotizing fasciitis, pemphigus, ne, dermatoinyositis, granuloma, lymphoma, sepsis, aplastic anemia, multisystem organ failure, multiple myeloma, monoclonal gammopathy of unknown icance, chronic inflammatory demyelinating polyradiculoneuropathy, inflammatory myopathies, thrombotic thrombocytopenic pui’pura, is, , sia, hemolytio anemia, encephalitis, myelitis, myelopatliy especially associated with human T—cell lymphotropic virus—1, leukemia, multiple sclerosis and optic neuritis, asthma, epidermal necrolysis, LambertnEaton niyastlienic syndrome, neuropa‘rhy, uyeitis, Guillain- Barre syndrome, graft versus host disease, stiff man syndrome, oplastio cerebellar degeneration witlt anti—Yo antibodies, paraneoplastic alomyelitis and sensory neuropathy with antimHu antibodies, systemic vasculitis, systemic lupus matosus, autoimmune diabetic neuropathy, acute idiopathic dysautonomic neuropatliy, Vogt—Koyanagi-Harada Syndrome, niultifocal motor neuropatliy, lower motor neuron syndrome associated with anti—/GMl, demyelination, membranoproliferative glomerulonephritis, cardiomyopatliy, Kawasaki's disease, rheumatoid arthritis, and Evan's syndrome, CIDP, MS, derntatomyositis, muscular dystrophy. The term "intravenous administration” as used herein includes all techniques to deliver a compound or composition of the present invention to the systemic ation Via an intravenous injection or infusion. {(38143} Dermal gel, lotion, cream or patch: vitiligo, Herpes zoster, acne, clielitis. {90144} Rectal suppository, gel, or infusion: ulcerative colitis, heinorrlioidal inflammation.

{Gill 5} Oral as pill, troche, encapsulated, or with c coating: Crohn's disease, celiac sprue, irritable bowel syndrome, inflammatory liver disease, Barrett’s gus. {(130146} lntra-cortical: epilepsy, Alzheimer's, multiple sclerosis, Parkinson's Disease, Huntington's Disease. 7} lntra~abdominal infusion or implant: endometriosis. {@9148} Medical devices: coated on ry artery , prosthetic joints. firerapem’ic Aguilieaiiom offlpfimaliy M'annfocmred GL~2045 [Annotation] yzus [Annotation] yzus {89149} in one embodiment, a method for treating or preventing a disease or condition such as an autoimmune disease, inflammatory disease, or complementumediated disease or ion is provided. {lllllfitll Based on rational design and in vitro and in viva validations, the optimally manufactured GLn2045 of the present ion will serve as an ant biopharmaceuticals for treating inflammatory diseases and disorders, as well as for altering immune on in a variety of other contexts such as bioimmunctherapy for allergies, cancer, autoimmune diseases, infectious es, and inflammatory diseases. l conditions suitable for treatment with the immunologically active optimally manufactured (EL—26.345 disclosed herein include any disease caused by or associated with complement activation or complement—mediated effector functions, including sed or inappropriate ment activity. Such medical conditions e those that are tly or have previously been d with complement binding drugs such as eculizumab. Eculizumab binds to complement protein C5 (a complement protein that is downstream of Cl and Clq in the classical complement pathway), inhibiting its cleavage and subsequent complement—mediated cell lysis, The biomiinetics of the t invention provide a safe and effective alternative to other compleinent—hinding drugs known in the art. For example, in some embodiments, the biomimetics of the present invention bind Cl o, the first subunit in the Cl complex of the classical complement pathway. l conditions le for treatment with the immunologically active optimally manufactured stradomers include, but are not limited to, myasthenia gravis, hemolytic uremic syndrome (l-lUS), atypical hemolytic uremic syndrome (allUS), smal nocturnal hemoglobinuria (Phil-l), membranous nephropathy, neuromyelitis optica, antibody—mediated ion of allografts, lupus nephritis, macular degeneration, sickle cell disease, and membranoproliterative glomerulonephritis (MPGN), Additional medical conditions suitable for treatment with the immunologically active optimally manufactured GL_2045 described herein include those currently routinely treated with broadly immune suppressive therapies including hl‘v'lG, or in which lilVlG has been found to be clinically useful such as autoimmune cytopenias, chronic inflammatory demyelinating polyneuropathy, Guillain—Barre’ syndrome, myastlienia gravis, anti~Factor Vlll autoimmune e, dermatomyositis, vasculitis, and uveitis (See, F. G. van der Meche et al., N Engl. J} Med. 326, 1123 (19.92); P. Gaidos er al, Lancet i, 406 (1984}; Y. Sultan, M. et al, Lancet ii, 765 (l984); M, C. Dalakas or £11,, N. Engl. J. Med. 329, l993 (1993); l). R. Jayne et al., Lancet 337, 1137 (1991); l). LeHoang, et al., Ocul. lmmunol lnf’lamm. ation] yzus [Annotation] yzus 8, 49 (2000)) and those cancers or inflammatory disease conditions in which a monoclonal antihody may he used or is already in clinical use. Conditions included among those that may be effectively treated by the compounds that are the subject of this invention include an inflammatory disease with an imbalance in cytokine networks, an autoimmune disorder ed by enic autoantibodies or autoaggressive T cells, or an acute or chronic phase of a chronic relapsing autoimmune, inflammatory, or infectious disease or process. {30151} In addition, other medical conditions having an inflammatory component involving complement will t from ent with the GLnZOKlS composition such as , lupus erythematosus, glomerulonephritis, glomerular nephropathy, arthritis, autoantihody—mediated diseases ing autoimmune tic anemia and autoimmune heart disease, multiple sclerosis, Amyotrophic Lateral Sclerosis, Huntington‘s Disease, Alzheimer's Disease, Parkinson’s Disease, Inflammatory Bowel Disease, paroxysman nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, ischemia—reperfusion injuries including as examples myocardial infarction, spinal cord injury, and , rejection of transplanted organs or blood, Hepatitis B, Hepatitis C, Human Immunodeficiency Virus ated inflammation, adrenoleulcodystrophy, and epileptic disorders ally those believed to be associated with postviral alitis including Rasmussen Syndrome, West Syndrome, and oX—Gastaut Syndrome {00152} The l approach to therapy using the Clio-2045 composition described herein is to administer to a subject having, a disease or ion, a therapeutically effective amount of the GL-2045 composition to effect a treatment. In some embodiments, diseases or conditions may be y categorized as inflammatory diseases with an imbalance in cytolrinc networks, an autoimmune disorder ed by pathogenic autoantihodies or autoaggressive T cells, or an acute or chronic phase of a chronic relapsing disease or process. {@9153} The term ”treating” and “treatment" as used herein refers to administering to a subject a therapeutically effective amount of an optimally manufactured stradomer of the t invention so that the subject has an improvement in a disease or condition, or a symptom of the disease or condition. The improvement is any improvement or remediation of the disease or condition, or symptom of the disease or condition. The improvement is an observable or measurable improvement, or may be an improvement in the general feeling of eing of the subject. Thus, one of skill in the art realizes that a treatment may improve the disease condition, but may not he a complete cure for the disease. Specifically, improvements in subjects may include [Annotation] yzus [Annotation] yzus one or more of: decreased inflammation; decreased inflammatory laboratory s such as C— ve n; sed autoimmunity as ced by one or more of improvements in autoimmune markers such as autoanti‘oodies or in platelet count, White cell count, or red cell count, decreased rash or purpura, decrease in weakness, numbness, or tingling, increased glucose levels in patients with hyperglycemia, decreased joint pain, inflammation, swelling, or ation, se in cramping and diarrhea frequency and volume, decreased angina, decreased tissue inflammation, or se in seizure frequency; decreases in cancer tumor burden, increased time to tumor progression, decreased cancer pain, increased survival or improvements in the quality of life; or delay of progression or improvement of osteoporosis. {90154} The term “therapeutically effective amount” as used herein refers to an amount that results in an improvement or remediation of the symptoms of the disease or ion. One of ordinary skill in the art will understand that the therapeutically effective amount of the GL~2045 produced herein can vary depending on the final drug substance. Thus, for example, if one were to eliminate all lower order multimers, it is conceivable that a reduced dose of the resulting higher order multiniers may be required. As such, there is more than one ”therapeutically effective dose” of GIL—2045. {till 155} As used herein, ”prophylaxis" can mean te prevention of the symptoms of a disease, a delay in onset of the symptoms of a disease, or a lessening in the severity of subsequently developed disease symptoms. {30156} The term ”subject" as used herein, is taken to mean any mammalian subject to which optimally manufactured stradomers of the present ion are administered according to the methods described herein. In a specific embodiment, the methods of the present disclosure are ed to treat a human subject. The methods of the t disclosure may also he employed to treat non-human primates (cg, monkeys, baboons, and chimpanzees), mice, rats, hovines, , cats, dogs, pigs, rabbits, goats, deer, sheep, ferrets, gerbils, guinea pigs, hamsters, hats, birds {at}, chickens, turkeys, and ducks), fish, and reptiles to produce species—specific or chimeric stradomer molecules. {89157} Complement inhibition has been demonstrated to se antibody—mediated diseases (See for example Stegall at all, American l of Transplantation 2011 Nov; ll(l):2405—2413_Epuh 20“ Sept 22). The optimally manufactured stradomers of the present invention may also be used to treat a disease or condition that is antiliodyuniediated. Auto— [Annotation] yzus [Annotation] yzus antibodies e many known autoimmune diseases and likely play a role in numerous other autoimmune diseases. Recognized antibody mediated diseases in which the optimally manufactured mers of the present ion may be used include, but are not d to, anti" glomerular basement ne antibody mediated tis including Goodpasture’s; antiudonor antibodies (donorsspecific alloantibodies) in solid organ transplantation; antinAquaporinnél dy in neuromyelitis optica; anti—VGKC antibody in neuroniyotonia, limbic encephalitis, and Mowan’s syndrome; icotinic acetylcholine receptor and anti—MuSK antibodies in myasthenia grayis; anti"VGCC antibodies in Lambert Eaton myasthenic syndrome; antinAMPAR and anti~GABA(B)R antibodies in limbie encephalitis often associated with ; anti—GlyR antibodies in stiff person syndrome or hyperekplexia; anti—phospholipid, anti~cardiolipin, and anti— §32 glycoprotein 1 antibodies in recurrent spontaneous abortion, Hughes syndrome, and systemic lupus erythematosus; lutamic acid decarboxylase antibodies in stiff person syndrome, autoimmune cerebellar ataxia or limbie encephalitis; anti-NMDA receptor antibodies in a newly- described me including both limbic and tical features with prominent movement disorders often in young adults and children that is often associated with ovarian teratoma but can be non—paraneoplastic; anti~double stranded DNA, anti—single stranded DNA, anti~RNA, anti~SM, and anti—Clo antibodies in systemic lupus erythematosus; anti—nuclear and anti~nucleolar antibodies in connective tissue diseases including scleroderma, Sjogren’s syndrome, and polyinyositis including anti-Re, anti—La, anti—Sol 70, anti—Jed; anti—rheumatoid factor dies in rheumatoid arthritis; anti-hepatitis B surface antigen antibodies in polyarteritis nodosa; anti— centroniere antibodies in CREST syndrome; anti—streptococcal antibodies in or as a, risk for endocarditis; anti-thyroglobulin, anti-thyroid peroxidase, and anti-TSl-l receptor antibodies in l-lasliinioto‘s thyroiditis; anti-Ul RNP antibodies in mixed connective tissue disease and systemic lupus erythematos us; and anti-desinoglein and anti~l<eratinocyte dies in pemphigus. {(130158} The (EL—2045 composition of the present invention may be used to treat conditions including but not limited to congestive heart failure {Cl-{F}, vasculitis, rosacea, acne, eczema, myocarditis and other conditions of the myocardium, ic lupus erythematosus, diabetes, lopathies, synoyial fibroblasts, and bone marrow strorna; bone loss; l’aget‘s e, osteoclastonia; multiple myeloma; breast cancer; disuse osteopenia; malnutrition, periodontal disease, r’s disease, Langerhans cell histiocytosis, spinal cord iniury, acute septic arthritis, osteomalacia, Cushing’s syndrome, monoostotic fibrous dysplasia, polyostotic fibrous dysplasia, ation] yzus [Annotation] yzus periodontal reconstruction, and bone fractures; sarcoidosis; osteolytic bone cancers, lung , kidney cancer and rectal cancer; bone metastasis, bone pain management, and humoral malignant hyperealcemia, ankylosing spondylitis and other spondyloarthropatliies, transplantation rejection, viral infections, hematologic neoplasias and neoplastic—like conditions for example, l-lodgkin’s lymphoma; non—Hodgkin's lymphomas ittls ma, small lymphocytic lymphoma/chronic lymphocytic leukemia, mycosis fungoides, mantle cell lymphoma, follicular lymphoma, diffuse large B-«cell lymphoma, marginal zone lymphoma, hairy cell leukemia and lymphoplasmacytic leukemia), tumors of lymphocyte precursor cells, including Bncell acute lymphoblastic leukemia/lymphoma, and T—cell acute lymphohlastic leukemia/lymphoma, thymoma, tumors of the mature T and NE: cells, ing eral T—cell ias, adult '1‘— cell leukemia/T—cell lymphomas and large granular lymphocytic leukemia, Langerhans cell histiocytosis, myeloid neoplasias such as acute myelogenous leukemias, including Alt/{L with maturation, Ah .1 without differentiation, acute promyelocytic leukemia, acute niyelornonocytic ia, and acute monocytic leukemias, niyelodysplastic syndromes, and chronic myeloproliferative disorders, including chronic myelogenous leukemia, tumors of the central nervous system, eg, brain tumors (gliorna, neuroblastonia, astrocytoina, medulloblastoma, ependyrnoma, and hlastonia), solid tumors (nasopliaryngeal cancer, basal cell carcinoma, pancreatic cancer, cancer of the bile duct, Kaposi's sarcoma, testicular cancer, uterine, vaginal or cervical s, ovarian cancer, primary liver cancer or endometrial cancer, tumors of the vascular system sarconia and hemangiopericytonia» or other cancer. {(38159} The Gl_..~2045 composition of the present ion may be used to treat autoimmune diseases. The term ”autoimmune e” as used herein refers to a varied group of more than 80 es and conditions. in all of these diseases and conditions, the underlying problem is that the body‘s immune system attacks the body itself. mune diseases affect all major body s including connective tissue, nerves, muscles, the endocrine system, skin, blood, and the respiratory and gastrointestinal systems. Autoimmune diseases include, for example, chronic inflammatory demyelinating polyneuropathy, multifocal motor neuropathy, systemic lupus erythematos us, rheumatoid arthritis, multiple sclerosis, myasthenia gravis, and type 1 diabetes. {89169} The disease or condition ble using the compositions and methods of the present invention may be a hematoimmunological process, including but not limited to sickle cell [Annotation] yzus [Annotation] yzus e, idiopathic thrombocytopenic purpura, alloimmune/autoinimune thromhocytopenia, acquired immune bocytopenia, autoimmune neutropenia, autoimmune hemolytic anemia, parvovirus Blg—associated red cell aplasia, acquired antifactor Vlll autoimmunity, acquired yon ‘Willebrand e, multiple myelorna and monoclonal gammopathy of unknown significance, sepsis, aplastic anemia, pure red cell aplasia, Diamond~Blackfan anemia, hemolytic e of the newborn, Immune—mediated neutropenia, refractoriness to platelet transfusion, al, post- transfusion purpura, hernolytic uremic syndrome, systemic vasculitis, thrombotic throm‘bocytopenic purpura, or Evan’s syndrome. {99161} The disease or condition may also be a neuroimmunological process including, but not limited to, GuillainnBarre syndrome, chronic inflammatory demyelinating polyradiculoneuropathy, paraproteinemic lgM demyelinating polyneuropathy, Lambert—Eaton myasthenic syndrome, myasthenia grayis, multifocal motor athy, lower motor neuron syndrome ated with anti—GMl , demyelination, multiple sclerosis and optic is, stiffman syndrome, paraneoplastic cerebellar degeneration with anti—Yo dies, paraneoplastic encephalornyelitis, sensory neuropathy with anti—Hi: antibodies, epilepsy, encephalitis, myelitis, myelopathy especially associated with human. T-cell lymphotropic virus- 1, autoimmune diabetic neuropathy, Alzheimer’s disease, Parkinson’s disease, Huntingdon’s disease, or acute idiopathic dysautonomie neuropathy, {(36162} The e or condition may also be mation or autoimmunity associated with hearing loss or vision loss. For example, the disease or ion may be autoimmun e-related hearing loss such as noise—induced hearing loss or age—related hearing loss, or may be associated with implantation of devices such as hearing s (e.g, cochlear implants). In some ments, the compositions provided herein may he administered to a subject prior to, concurrently with, or subsequent to the implantation of a device. {(130163} The disease or condition may also he a rheumatic disease s including, but not limited to, Kawasalri‘s disease, rheumatoid arthritis, Felty‘s syndrome, ANCA—positive itis, spontaneous polymyositis, dermatomyositis, antiphospholipid syndromes, recurrent spontaneous ahortions, systemic lupus erythematosus, juvenile idiopathic tis, Raynaud‘s, CREST syndrome, or uvei'tis. {89164} The disease or condition may also he a dermatoimmunological disease process including, but not limited to, toxic epidermal necrolysis, gangrene, granuloma, autoimmune skin [Annotation] yzus [Annotation] yzus blistering diseases including pemphigus Vtilgaris, hullous pernphigoid, peinphigus foliaceus, vitiligo, Streptococcal toxic shock syndrome, derma, systemic sclerosis including diffuse and limited cutaneous systemic sclerosis, or atopic dermatitis ially steroid dependent). {(130165} The disease or condition may also he a musculoskeletal immunological disease process including, but not limited to, ion body myositis, necrotizing fasciitis, inflammatory myopathies, myositis, anti—decorin (Bl antigen) myopatliy, paraneoplastic ic myopathy, X— linked vacuolated myopathy, penacillamine—induced polymyositis, atherosclerosis, coronary artery disease, or cardiomyopathy. {99166} The disease or condition may also be a gastrointestinal logical e process ing, but not limited to, pernicious anemia, autoimmune chronic active hepatitis, primary biliary cirrhosis, celiac disease, itis lierpetiformis, cryptogenic cirrhosis, reactive arthritis, Crohn‘s disease, Whipple's disease, ulcerative colitis, or sclerosing cholangitis, {$0167} The disease or condition may also be graft versus host disease, antibody—mediated rejection of the graft, one marrow transplant rejection, post—infectious disease inflammation, lymphoma, leukemia, neoplasia, asthma, Type 1 Diabetes mellitus with anti-beta. cell antibodies, Sjogren's me, mixed connective tissue disease, Addison’s disease, Vogt~Koyanagi—Harada me, membranoproliferatiye glomerulonephritis, Goodpasture's syndrome, Graves' disease, Hashimoto's thyroiditis, Wegener’s granulomatosis, mieropolyarterits, Churg~Strauss syndrome, polyarteritis nodosa, or inultisystem organ failure {00168} “Allergy,” as used herein, includes all immune ons ed by lgE as well as those reactions that mimic lgE—niediated reactions. Allergies are d by ens, ing proteins, peptides, carbohydrates, and combinations thereof, that trigger an lgE or 'lng—lilre immune response. ary allergies include nut allergies, pollen allergies, and insect sting allergies. Exemplary allergens include urushiol in poison ivy and oak; house dust antigen; birch pollen components Bet V l and Bet v 2; the 15 lei) antigen in celery; apple antigen Mal d l; Pru p3 in peach, 'l‘imothy grass pollen allergen l’hl p 1; Lol p 3, Lol p l, or Lol p V in Rye grass; Cyn d l in Bermuda grass; dust mite allergens dust mite Der pl Der p2, or Der fl; d-gliadin and y—gliadin epitopes in gluten; bee venom phospholipase A2, Ara h l, Ara h 2, and Ara h '3 epitopes in peanuts. {00169} In another embodiment, the (EL—2045 composition described herein could be ed in a priming system wherein blood is drawn from a t and transiently contacted with the optimally manufactured stradomer(s) for a period of time from about one half hour to about [Annotation] yzus [Annotation] yzus three hours prior to being introduced back into the t. in this form of cell therapy, the patient's own effector cells are exposed to the optimally manufactured stradomer that is fixed on a matrix ex vivo in order to modulate the effector cells through exposure of the effector cells to the lly manufactured stradomer. The blood, including the modulated effector cells, is then infused back into the patient. Such a priming system could have us clinical and eutic applications. {@0179} The GLnZtl45 composition disclosed herein may also be readily applied to alter immune system responses in a variety of ts to affect specific changes in immune response profiles. Altering or ting an immune response in a subject refers to increasing, decreasing or changing the ratio or components of an immune response. For example, cytokine production or secretion levels may be increased or decreased as desired, by targeting complement along with the appropriate combination of Felts with a stradomer designed to bind complement and interact with those receptors. Antibody production may also be increased or decreased; the ratio oftwo or more cytokin es or immune cell receptors may be changed; or additional types of eytokines or antibodies may be caused to be produced. {(39171} in a preferred embodiment, a subject with an autoimmune or inflammatory disease has their immune response altered comprising, the step of administering a. therapeutically effective amount of the 5 composition described herein to a subject, wherein the therapeutically effective amount of the GL~2®45 composition alters the immune response in the subject. Ideally, this intervention treats the disease or condition in the subject, The altered immune response may be an increased or a decreased response and may involve d cytoltine levels including the levels of any of lL-lRA and other lL—l family members, lL—6, lL—lO, ill—3, lL— 3 lL—7, lL—Ll, lls~ 12, lL—l3, ll_»l 7, lL-l receptors, TNF—a, other TNF family members and TNF receptors, lFN-a, other interferon family members and interferon receptors or ine levels including the levels ofany of the CCL, CXC, KC, and FAMl 9 cheinokine family members. in a preferred embodiment, lL-6 or lL—S is decreased in response to therapy. In an especially red embodiment, lL—6 and lb?» are decreased in se to therapy and/or lL—lO or lL—lRA are increased in response to therapy. The invention is, however, not limited by any ular mechanism of action of the described hiomimetics. The altered immune se may be an altered tibody level in the subject. The altered immune response may be an altered autoaggressive ll level in the subject. {@9172} For example, reducing the amount of 'l7NFmalpha production in autoimmune diseases can have therapeutic effects. A practical application of this is anti— alpha antibody [Annotation] yzus [Annotation] yzus therapy (eg; REMICADECE), which is clinically proven to treat plaque psoriasis, rheumatoid arthritis, tic arthritis, Crohn’s Disease, ulcerative colitis, and ankylosing spondylitis. These autoimmune diseases have distinct etiologies but share key immunological components of the disease processes related to inflammation and immune cell ty. A stradomer designed to reduce 'I‘Nlilalpha production will likewise be effective in these and many other autoimmune es. The altered immune response profile may also be direct or ct modulation to effect a reduction in antibody production, for example tibodies ing a subject’s own tissues, or altered auto—aggressive ’l‘~cell levels in the suhiect. For example, multiple sclerosis is an autoimmune disorder involving autoreactive T—cells which may he treated by interferon beta therapy. See, eg, Zafranskaya M, at all, Immunology 2007 May; :29—39—Epub 2006 Dec l8.

An optimally manufactured stradomer designed to reduce autoreactive "if—cell levels will likewise be effective in multiple sclerosis and may other autoimmune diseases involving autoreaotive T“ cells. {not ’73} The (”Ills—2045 composition described herein may he used to modulate expression of co—stimulatory molecules from an immune cell, including a tic cell, a macrophage, an osteoclast, a monocyte, or an NK cell or to inhibit in these same immune cells’ differentiation, maturation, or cytolcine secretion, including interleukin~l2 (lL- l2), or to increase cytokine secretion, including interleukin—l0 (ll,~ l0), interleukin—661,6), or lLl ~RA, A skilled artisan may also validate the efficacy of an optimized logically active biomimetic by exposing an immune cell to the optimized immunologically active hiomimetic and measuring modulation of the immune cell funoti on, wherein the immune cell is a dendritic cell, a macrophage, an last, or a monocyte. in one embodiment, the immune cell is d to the optimized immunologically active hiominietic in virro, further comprising the step of determining an amount of a cell surface receptor or of a cytokine production, wherein a change in the amount of the cell surface receptor or the cytoliine production indicates a modulation of the immune cell function. in another embodiment, the immune cell is exposed to the optimized immunologically active hiomimetic in vivo in a model animal for an autoimmune disease, further comprising a step of ing a degree of improvement in the autoimmune e. {00174} The (EL-2045 composition described herein may also he used as a component of a device. ln some embodiments, the GL—ZOdS provided herein may be coated on a device, such as a medical implant. For example, the optimally ctured stradomers may be coated on a [Annotation] yzus ation] yzus coronary stent or as part of nanopartlcle therapy to enhance penetration and prolong drug release, for example for intra—ophthalrnic use in uveitis or macular degeneration. The optimally ctured stradorners described herein may also be used as a component of a diagnostic. in some embodiments, a skilled n may alize therapy by determining, in which patients, use of a stradonier may be particularly beneficial. For example, the skilled n may expose a patient 3 immune cells to the immunologically active bionnmetic and measure modulation of the immune cell’s activation or maturation by flow cytometry or cytokine profile in order to identify high responders. {99175} All references cited herein are incorporated by reference in their entireties.

EXAMPLES {@9176} Various approaches in manufacturing process were taken to optimize the combination of high protein titer, long Viability with concomitant low cellular debris, and production of higher—order multimers of Git—2045. ically, the ing aspects of the upstream manufacturing s were varied to determine the optimal conditions for Gl_,—20-i15 products with the property of increased. erization: basal media, type of feed, timing. of feed, temperature shift, aeration, and shake flask conditions In each instance, cell density, viability, protein titer, and n‘iultimerization were analyzed in order to identify optimal conditions. Further, aspects of the downstream manufacturing process, including buffers, wash protocols, and column selection, were varied to determine the optimal conditions for purification and filtration of» GL— 2045 wherein the optimal n'iultimerization profile of (314—2045 was maintained. The following examples are provided by way of illustration only and not by way of limitation.

Exam le 1 —~ Fractionation and Biolayer interferometry anal. sis of Gthlld‘S {80177: Solutions of (EL—2045 were fractionated using a GE l-li—Load 26/60 Superdex 200 pg column (GE, #l7ulO7lufll) in 005 Ml TrisullCL + 0. l 5 M NaCl buffer (pl—l 7.5). 3.2 mL Glow 2045 on was loaded at a flow rate of 2.6 mL/min, Six fractions (1—6) were ted in 240 mL volumes and n tration determined lay UV measurement at 280 nm (Fifi. 1A).

Multimerization for each of the 45 fractions was assessed. Briefly, samples of each of fractions l—6 were loaded onto 4—129/éinon—reducing Nu—Page 8T gels (invitrogen, #NPO3ZZBOX).

Samples were run for approximately 3 hours at 150 volts. Results are provided in and [Annotation] yzus [Annotation] yzus trate distinct differences in the presence of higher order multimers of 5 between Fractions lube Fractions l~3 are comprised of lower order multimers (ewg. bands 1-4). Fraction l is comprised nearly exclusively of homodiiner having an apparent MW of 55KB l:Band l, MW estimations from non—reducing SDS~PAGE). Fraction 2 is sed of approximately 97% dimer of the homodimer having a MW of 110 Kl) (Band 2). Fraction 3 is comprised primarily of Bands 3 and 4 having MWS of 165 Kl) and 220 KB, respectively along with smaller amounts of Bands 2, 5 (NFW = 275 K0) 6 (lVlVV = 330 KB) and 7 {ll/l‘i/V = 385 KB) Fraction 4 is comprised predominantly of bands 4, 5, and 6,, along with smaller amounts of Bands 3, 7,, S (h/IW’ = 440 Kill) and higher order bands. However, Fractions 5 and 6 are comprised predominantly of higher order multimers {bands 5+). {00178} Fractions of 4.5 were analyzed for binding to FclellA receptor using a biolayer interferometry kinetic binding is. Biolayer interferometry detects the binding between a ligand immobilized on the biosensor tip surface and an analyte in solution. When g occurs it es an increase in optical thickness at the biosensor tip, which results in a wavelength shift (detected as a response unit of “RU”). The maximum g level (RU max) is the maximum possible amount of sample binding at equilibrium that saturates the amount of ligand on the sensor e. {00179} His—tagged, receptor proteins (5 rig/’mL) were bound to an anti-His sensor tip (Anti- Penta—l-iis l-llSlK, ForteBio Cat. # 18—5121) in lX kinetic analysis buffer from ForteBio (Cat. # 18—1092) for 300 seconds. The loaded sensor was transferred into 1X ltinetic buffer t labeled receptors or ligands in order to obtain baseline measurements for 60 seconds. After obtaining a baseline, the on rate of the receptor/protein was measured by transferring the sensor tip to a la: kinetics buffer containing the purified stradomer of choice for 60 seconds at concentrations of 50 llg/n’lL, 25 ng/mL, and 12.5 {lg/hi. Off rate was measured for 300 seconds by erring the sensor tip to a lX kinetics buffer, and RU value, on rate value, dissociation rate and Kd value was calculated using the ForteBio software. {00180} Binding curve results are shown in and kinetic binding data calculated by ForteBio Octet re is provided in Table 2. These binding curves trate higher avidity with an increasingly lower off rate for fractions containing higher molecular weight GL—Ztltli {e.g., ons 3, 4, 5, and 6) than observed for lower molecular weight fractions tag. Fractions 1 and [Annotation] yzus [Annotation] yzus 2), and indicate that the high molecular weight fractions of 45 hind more avidly than the lower lar weight fractions.

Tahle 2: Summary ol Kinetic Binding Data for GL—2ll45 Fractions ' '""" ' (ingots ' i 23213-07 looms .33E-02 33430:} 00343 413-10 2"le 01m 4 ‘iZ>-= 02782 n 1366 Exam le 2 — Com lenient—dc endent Cell CDC Killino Assav with GL~2045 Fractions } The ability of GL~2045 fractions to inhibit complement activation was assessed.

{EL-2045 was fractionated by size ion chromatography in to 6 fractions (), each analyzed for inultiinerization on a non—reducing gel (HG, 3B) To determine the effects ot‘ each fraction on ment tion, CDZO—expressing Will—2 cells were incubated with an anti— CD20 monoclonal antibody for 20 minutes, after which the cells were centrifuged and re- ded in fresh media Cells were then incubated in a 96 well plate in media containing each of the ons 1—6 described herein as well as unfractionated Git-2045 as comparison at one of six concentrations; lOO tig/inL, 50 tig/mll, 20 gig/niL, l0 tig,"rnl_.., 5 ttg/rnl_,, or l rig/into Serum was added to the cell suspensions in order to initiate complement ent cell lysis, and the plate was incubated at 37° C for} hours Cell death was quantitated with the Promega Cytotox Glo Assay, The Cytotox Assay Reagent was added to each well of the plate, and the plate was incubated in the dark for l5 minutes at room temperature. The scence after l 5 minutes was read on a Promega GloMax luminoineter and cell death was calculated from this reading. Results are shown in —4D and demonstrate that fractions 5 and 6 (containing the higher molecular weight rnultirners in bands 543) showed more profound inhibition of CDC than the smaller molecular weight multimers present in fractions l—4. It is also noted that only fractions that comprise hand 4 and higher demonstrate effective inhibition of CDC, tent with the polyvalent Fc binding of higher order inultiiners to hexanieric- Cl q.

Examgle 3 — Binding of Gig—2945 Fractions to FcIRll'la [Annotation] yzus [Annotation] yzus {001821 The binding of GL—ZOdS ons to Fey/Killer was determined. GIL-2045 l l-003L’l—95) with a binding buffer of 20 mM sodium pl’iosphate, 015 M NaCl, pH 7.2 and was eluted with 0.1 M glycine, pH 2.7 (). Affinity chromatography~purified (EL—2045 was stored in 1X PBS, pH 7.2 ty Biological, .lnc #119—069—101). A pool of ed GIL—2045 was further dialysed against 50 mM Sodium Acetate, pH 5.0 and polished by cat~ion exchange chromatography on a POROS ClEX column (GOPURE Column 1.2 cm D x 10 cm L, Pores XS Life Technologies, #4448885) with a binding buffer of 50 mM Sodium Acetate pH 5.0 and an elution gradient (0 to 100% elution ) with 50 mM Sodium Acetate, 1 M NaCl, pH 5.0. This polishing step was performed without the elimination of the highest order niultiniers and/or unu ordered aggregates from the final fractions As a final step, the ClEXupolished GL~2045 was concentrated to a volume of E 5 niL, and buffer exchanged against the gel filtration running buffer and injected into the gel filtration column (liliload 26/60 Superdex 200pg {GE # 17—1071~01)) using 0.05 M 'l‘ris-HCl + 0.15 M NaCl, pH 7.5 as running buffer (Tris HCL, pH 7.5 '1‘eknova #511075). Fractions were then analyzed for rnultimerization by gel analysis ( and F1G. SC). {00183} Binding of fractionated {EL—2045 to Fclella was determined using an Felella ELISA binding assay. y, 96 plates were coated with recombinant Felella and d to react with (EL-2045. After washing, the amount of Pclella-bound material was determined using an Fe detecting mAb in an ELISA based assay ( and, ). ECso values for each on are shown in Table 3. These results demonstrate that higher order multimers ions 1C4, 1C5, 1C6, 1C7, U38, 1C9, lC10, and 1011) demonstrate more avid binding, noted by a low 13350, to Pct/Rllla than lower order niultimers (Fractions 1139, 1E4 and 1F7) and, surprisingly, more avid binding than the highest molecular weight rnultimers (Fractions 1C3, lCZ, lCl, 181.2, 1181 1. The very highest molecular weight fractions are presumed to comprise some lower y high molecular weight aggregated fractions along with the highly functional highest order multimers (ag Fractions 11811, 11312, lCl, 1C2, and 1C3). These results indicate very surprisingly that not all high molecular weight ons of GL~2045 demonstrate increased binding to FcyRs. likely due to effects of unordered aggregation of the homodimer as opposed to the formation of highly—ordered, high molecular weight rnultimers. These data te the need for optimized downstream manufacturing methods (including optimized conditions for protein A purification, ion exchange tography, and hydrophobic interaction chromatography) in [Annotation] yzus [Annotation] yzus combination with the optimal upstream manufacturing methods to result in the preductinn and retention of highurnnlecular weight, higher order inultiiners and ation of unuordered, high meiecular weight aggregates (ag, lBll and 1812), which are less effective at g target low" affinity recepters (see 136350 value for 181} and 11812 in Table 3).

Table 3: EEC-39 Values fer Gig—2945 Fractions Binding tn FelelHa "'i7"i3iiZil57fi"'“EXEZEEEX""""""" rzcse 11 mil 1:89 NT irii 1.60 1134 2.26 1135 NT 1139 3.15 1134 3.82 1138 NT 11?? 15.7 1F8 NT NTr Not Tested Exam le 4 —~ (Ea—induced chernetaetle anal sis of GLuZMS fractions 84} (EL—2045 cell culture was grown in PowerCHQZ media (Lonza: # UZl—O’ifi) with L—Glutamine {Lonza, # l7—605E) and HT supplement (Life Technelogies, # l1067—G30). (3194045 supernatant was purified by affinity chromatography with n A I'IiTrap MabSelect Sure (GE, 9% l l~0034—95) then fractionated with AIEX en Q FF (GE, # 28495054 0) using different pH conditions to separate the low molecular hands t0 the high molecular hands. Results are shown in [Annotation] yzus ation] yzus (GLuGLM~Ol recombinant, unfractionated Fc (LGOOl ), GL—GLM—OZ tionated GL— 2045, GL—GLM—OS fractionated GLuZOZlS at pH 6.0, GL—GLM—Oo fractionated (EL—2045 at pH {3.5, GLuGLM-G7 fractionated (EL—2045 at pH 7.0, /lullfi fractionated Cris—2045 at pl-l 7.5). Finally, the different fractions were concentrated and dialyzed against HESS (Lonza, # l0~ 5271?). {00185} GL—2045 atant was purified by affinity chromatography with protein A (pA) HiTrap MabSelect Sure (GE, #- ll~0034—95) with a binding buffer of 20 mM sodium phosphate, O.l5 M NaCl pH 7.2. After a first wash with the binding buffer and a second, wash a buffer comprising 1 M NaCl, 5 rnM EDTA, 2 M Urea, 501ml phosphate pH 70, the protein bound to pA was eluted with 0.1M glycine, pH 2.7. {90186} Affinity chromatography-purified (EL—2045 was stored in 1X PBS, pH 7.0 (Quality Biological, lnc #l 194369401), 4- batches of ed Gl_.-—2045 were further diluted (6X) with SOniM, Tris—HCL at pH 6.0, 6.5, 7.0 or 7.5 and purified by anion exchange chromatography on a HiScreen Q FF column with a binding buffer of 50 mM Tris—HCL pH 6.0, 6.5, 7.0 or 7.5 and eluted by gradient elution (0 to l00% elution buffer) with 50 mM Tris—HCL + l M NaCl at pH 6.0, 65, 7.0, 7.5. {till 187} These purified fractions were utilized to determine the effects of G 44045 fractions on neutrophil chemotaxis. Briefly, complement CSa was added as a chemoattractant to the lower well of a Boyden chamber at a concentration of lnM. Prior to addition to the Boyden chamber, neutrophils {final concentration 2.25 x l06 cells/mi, purified from whole blood from PBMCS) were pro—incubated with the indicated GL—2045 ons (0.024 G pg/rnL final concentrations gig/mic) or recombinant Fc control (Glh/LOGl, 6001) for 30 minutes. Cell, sions were then added to the upper well of the Boyden chamber and incubated for 25 minutes. Following the tion period, migrated populations were assessed by counting the number of cells in the lower chamber for each condition and a percent cheniotaxis for each condition was determined (.

No chemotaxis was observed for GLUGLM—OOl (recombinant Fe control, Glllll). Higher order niultiiners (Fractions GL—GLM—OUZ, GL~GLM—G05, —OOS, and GL~GLM—OQ7 comprising hands 5 — l3) demonstrated more avid inhihition of duced chemotaxis than lower order ers (Fraction GL—GLM-GO8 comprising hands 1 — 4). {90188} The data provided in Examples l—Al trate the enhanced efficacy of higher“ order inultimers on GleOZI-S. Based on the ahove data, am manufacturing protocols were LI: lJi [Annotation] yzus [Annotation] yzus tested in order to determine the l conditions for the specific production of higher—order inultimers (cg. bands 5+ in FIGS, 1, 3, 5, and 7) of GLn2045 in order to achieve maximum biological efficacy.

Exam le 5 Base Media Screenin in S reduction {90189} The purpose of this ment was to test the effect of a panel of basal medias on {ii—2045 protein titer, cell Viability, cell density, and GL~2045 multimeriza’rion. } 5 was grown in ProCHOS media (Lonza #lZ—766Q) with LnGlutamine (Lonza #17—605E) and. sodium hypoxanthine and thymidine (HT, Gibco #11067—039) in a shaker incubator at 37C and 5% C02, After oassaging, the cells were washed and inoculated in duplicate at ()5 x106 cells/’mL in Selected Media (shown in Table 4) into a 59 mL 'l'ube Spin. Eacli tube contained l0 mL of culture and was placed in a Kuhner brand maglev shaker tor at 370C, % C02, 80% humidity and lSO rpm rotation speed. At day 4, 8, and l0, one milliliter of sample was taken from each culture for measuring cell density, cell Viability and e level. Samples were centrifuged and supernatants were stored at +4°C ed Medias that did not list r—‘l mM L— Glutamine and lX sodium hypoxanthine and tliymidine were supplemented with luv—Glutamine and HT as components. Growth conditions for Selected Media are shown in Table I—‘l.

Table 4: Selected Media Growth Conditions Medium Manufacturer CD FoniCHQ Life’l‘eclmologies T042 (error/rites CD) Tammi (X611) Hyclone curt/mono l-chloue ADCF MAB Sigma/Aldrich BalanCD CHO Growth A EX—CELL co cue CHO—S-SFM 11 lliife'l'echuologies E'Xiii5iiiiri Eli'élilééilfiiiiégiéém care SFM/lCHO ClonaCell-CHO CD Stemcell Tech.

GE Healthcare HYQ SFX— GE Healthcare CHO LM Cell Vents CHO 2m Millipore [Annotation] yzus [Annotation] yzus ActiCHO P GE "96.1325???“gig_1___1_9_______________ Mill}??? (3b Hyhgdgma "irrigateattaia"““““ 5 go {(30191} The (EL—2045 cell cultures grown in selected rnedia were assessed for cell density, cell viability, protein titer, and percent 0f higher order multinrers. Cell y and cell Viability assessments were perferined by mixing cells with 'l'rypan Blue. Viable and dead cells were counted using a manual cell r. Data for Day 4 and Day 8 of culture are sliewn for cell density ( Table 5) and cell Viability (MG. 10, Table 6). Of the 19 media tested, the greatest cell density at day 4 was Observed with ActiCHO P, CHOMACS CD, and CD FertiCHO. By day 8, the trend in cell y was negative for all media except for ActiCHO P, BalanCD CEO, ExpiCHO, Cell V'ente CHO 210, and Cell Vente CHO 210, ed to day 4. Of the 19 media, the greatest cell Viability at day 8 tested was observed with AetiCHO P l‘ellewed lay ExpiCHO, Cell Vente CHO 110, and HYQ SEX—CHO LM, The only media of the l9 media tested to have a ve trend in cell viability frem Day 4 to Day 3 was ActiCHO P. ore, the only media which produces high cell density at Day 4 and dees not. have a, negative trend for cell density at Day 8 is ActiCHO Table 5: GL-Ellélfil’mdueing CHO Cell Density Medium gill/:14) 231/:L) CD FortiCHO ' Tit—42 (CHOMACS CD) Hyelone CDM-lCHO Hy clone ADCF MAB HO3 CD Ex—Cell CH05 ActiCl-IO P BalanCl) CHO Growth A ExpiCl-lO Expression SFM4CHO ClonaCellnCHO CD HYQ SFX-CHO LM Cell Vente CHO 210 [Annotation] yzus [Annotation] yzus Cell Verne CHO 110 CD doma CD OptiCl—IO PowerCHO-GS Table 6: GL—ZfldS—Pmduelng CEO Cell Viability nay 4 Day 8 (savanna) (% Viable) CD Fen-leer) .

TC—42 genomes en) 99.4 on Hyclone cement) l-chloneADCFMAB ':. .1 .' PowerCi—im en Ex—Cell CHOS ActiCHOP EBBalanCD CHO Growth/3i EEXACELL CD (“H0 FxpiCHO F\pi”SCIOII :'sewcne ell—CHO Cl) HYQ SFX'—Cl-{O 11M: : .: Cell V'nto {31-10 :2 in Cell Vento CBC llO CD Hybridema CD OptiCHO HO-GS as2 :862 {90192} Cultures were spun down on day 10, filtered at 0.2 pm and kept at 4°C until purifieatinn using a pretein A affinity . For purifiea'tien, supernatant cultures were purified by affinity chromatography with 1 mL pretein A column HiIrap MahSelect SuRe (GEJ? ll 43034— 93) with a binding buffer of ZOmM sodium phesphate, (IISM NaCl, pH 7.2. The eelumn—bound protein was washed with the binding buffer fellewed by a seeend wash with l M NaCl, 5 mM EDTA, 2 M Urea, SOmM phosphate, pH 7.0. (EL—2045 bound to the column was eluted with 0.1 M e pH 2.7 and desalted in 1X PBS at pH 7.0 thrn Hil’rep 26/10 desalting column (GE, #l ’7— 5087—01). All samples purified were , at 40C. Measurements of protein titer were performed by biolayer interferometry (Octet) (FIG. II and Table 7). Of the 19 base media tested, the greatest ation] yzus [Annotation] yzus protein titer at day 10 was observed with Celt Vento (Li-10 1110 followed by BaianCD CHO Growth A Medium, ActiCI-K) P, and ExpiCHO. A significant drop in titer occurred with other media.

Tobie 7: {EL—2045 Titer Protein Medium CI) OptiCHO SFM4CHO PowerCI-{O—GS r042 (CHOMACS co) 93} in order to determine the percent of higher order multimers, each purified culture was run (in non—reduced form) onto an SDS—PAGE gel (NuPage 3—8945 Tris-Acetate. ge} Life Teohnoiogieg, it EAO3’7SZBOX). ,2 ug of protein was diluted in. 3 [AL of sample buffer (NuPage, LDS (4X), Life Teohnoiogies, # NPOOO7), 20 nM of iodoaeetamide (Bio Rad #1632109) and deionized (Di) water to a final volume of 10 .uL. Sampies were heated at 800C for ‘10 minutes. and ioaded onto the gei and run at 150V for E hour and 25 minutes using running buffer {Tris~Aeetate SDS (20X) (Life Technoiogies, # {4130041)}. Gele were washed in D1 water, stained with Simply/Blue Safe (Life Technologies, # [(36060) and destained in Di water After a ete ning a picture was taken using GEBOX system from e and the g, pattern was anaiyzed by densitometry with GeneToois, Syngene software The intensity in each individuai hand in each lane was measured (A and EEG. 12B).

[Annotation] yzus [Annotation] yzus {89194} Unexpectedly, the st percent of higher order multiniers above band 4 of the 19 media tested was observed with AetiCHO P ed by EXflELL CD (Tl-K) and (El-la S SFMZ (Table 8). These data te that increased percentage of higher—order inultirners is an independent variable to be controlled and is not simply correlated with an increase in total protein titer. AetiCHO P resulted in the third highest protein titer and greatest level of niultinierizatinri (45.9% of protein present in bands 5+), while Cell Vento CHO llO resulted in the greatest protein titer but a substantially lower level of multinierization (32.6% of protein present in bands 5+}.

Table 8: GL—2945 Mnltimerizaiinn Analysis ”/0 Bands % Bands Medium (14) ’54-) AciiCHOP tax-CELL CD CH0 CHO— sees/n l..iéseiigiiaieisa____________________________________________________________ H Velone ADC}? Cell Vento 2.10 Cell Vento lit) CD 0 acne ExiCHO CD H be‘ldOfiL’i Power CHO GS r, _: ._ Balance CD CHO crime Ciro Exam le 6 “it/ladle: Screenino‘ of S with feeds RecommendedFeeding Schedules {@195} Based on the s of the experiments in Example 5, four base media associated with the highest (EL—2045 protein titers and three base media associated with the lowest GL~2045 protein titers were subjected to a repeat experiment in which commercially ble feeds were provided during culture. Cell Vento ill) and ExpiCHQ which produced high titers, were not [Annotation] yzus [Annotation] yzus selected fur the feed experiment because no manufacturer—recommended feeds were fied.

{Tell Verito (ll-l0 110 is a te media to be used for cell adaptation without feeds, while Cell Ventu 2l 0 is used for culture in combination with feeds. Media and feed combinations used in this experiment are detailed in 'l‘ahle 9.

Table 9: Media Feed enmbinatluns Media Feed Cell Veirte CllO—Z 10 Cell Verne Feed—2 l0 Cysteine ./ ne Glucose EMT) Milli.ore #lOZSS31000 Fl‘vfl) Millicre # 024-88 BalanCD CHO Growth A Medium BalanCD CHE) Feed 1 lRVlNE #9] l 18 lRVlNE. #91127 CD FortiCHO CD EfficientFeed C AGT Nutriment Supplement Life Technolo ies #Al 1483—01 Life Tech. #A 13275-04 CDMAlCHU Cell Boost 4 (P8307) _liliziEl9£l§il§l1l§9§§§£l______________________________l:lllEl9£l§_§ll§ ________________________________________________________________________________ PowerCl-l03 CD Puwer Feed A L—Glutamine Lonza #12—772Q #8E02—0440 ADCF—Mah Cell Boost 4 (P8307) H clone #SH3034902 chlune #SH30857 AetiCl-lO 1" media Feed A, Feed, B FAA #lJ21~070 FAA #U 15—072, FAA #UOS—034’3‘ >Z‘PAA subsequently became part of GE Lifesciences {00196} (Ills—2045 clene 58 was ed in “PrcCHOS” media (Lcnza #lZ-766Q) with L— Glutamine {Loriza #1760513; and Sodium hypoxanthine and 'l‘liymidine (HT, Gihce til l06'7m030) in a shaker incubator at SW: and 5% C02. After passaging, cells were washed and inoculated at 0.5 xl 06 cells/ml, then sub~cultured when densities reach l X106 to '3>< l06 cells/niL and ,2: 80% Viability. Gil-2045 clone 58 was adapted directly intu selected media detailed in Table l0.

Adaptation was ered te when cells attained a stable dcuhling time (2.0 30 heurs) and a Viable cell density (VCD) ,2: 90% ever at least 2 3 passages. Cells were seeded at 0.5 x 105 cells/mL into selected media (d0) and incubated in a rd g rm in an incubator set at l50 rpm at 37C, 5% (:02, and high l’lltl’l’lldlly. For all culture except PowerCl-KB CD, feeding began on Day 3 (d3). Feeding for PewerCl—KB CD media began at dl. ”Fetal culture volume was 120 mL. Cultures were harvested when viability fell tu 50%. GLu2045 stable cell line was grown in each 0f 7 media along with the manufacturer recommended feed acccrding t0 the recommended protocol. The feeding strategy and schedule for each of the tested media is outlined in A and FIG: 138i [Annotation] yzus ation] yzus {@9197} Measurements of cell density, cell ity, and GL—ZOdS protein titer were performed throughout the study. For protein titer, samples were centrifuged to pellet cells and measurement of protein in cell atant was performed by biolayer interferometry (Octet) of the cell supernatant as described in Example 6. GL~2045 multimerization was assessed at termination of each arm of the study after protein A purification as described in Example 5.

Cultures were continued until day '14 or until Viability dropped below 599/0.

} Surprisingly, GL—2045 grown in ActiCHO P with cturerwrecommended feeds achieved a far higher peak cell density’ than (EL—2945 grown in any of the other media with manufacturer-recommended feeds. This superior cell density was surprisingly 3—fold or greater compared with all other media feed ations tested except for Cell Vento ill 0 (). {@9199} Further, GL.—2t‘:45 grown in ActiCHO P, on r‘omcno, or ADCF MAB with manufacturer—recommended feeds achieved far better (2-3 fold) cell viability at day it) than Glam 2045 grown in the other media tested with manuthcturer—reeommended feeds This demonstrates that these three media result in superior cell viability compared with l other media / feed combinations tested (FIG. l5). {oozes} onally, the GL~2045 CHO stable cell line grown in ActiCHO P media with manufacturer recommended ActiCHO Feed A and ActiCHO Feed 1% generated substantially higher titers than any other media, and manufacturer recommended feed tested (). The titer produced from the ActiCl-lO P culture was at least 4 fold higher than from any of the other media tested, ng 2 g/L at day 10 in shake flask compared to less than 500 n'ig/L for Cell Vento Cl-lO—2l O~ at day l2. and even less for other media. {90291} h/lultimerizati on of Gl_,-2045 as measured by percent multimer in bands 5 and above (5+) from the different culture conditions was determined as bed in Example 5 (NS. l7, Table 10). The highest rate of (EL—2045 niultimerization using inanut‘acturerurecommended base media and feeds was CD FortiCl—lO followed by ActiClrlO and PowerCl—KB. ADCE—Mab and BalanCD demonstrated significantly worse (EL—2045 multimerization compared with other culture conditions.

Table 1%: Peresnt multimers with Media + Feed combinations ----------------------------------------------------------------------------------------1'""""""""""'""""""""".

Media-+-Feed % % Bands Basins 1—4 5+ = [Annotation] yzus [Annotation] yzus (l) ADCF—Mah + Cell Boost 4 Feed 76.2% i 23.8% E "Eli'lié'iiééifilldi'Elli-"haliéliiééli'K"""""'l"ifii'idim’lEifi'iiiTl i3 (JDM4CHO+CellBoost4Feed 7 .s% E 25.2% l (4) co FortiCHO+ on Efficient Feed C 64.3% 35.7% (5) BalanCD CHO Growth A + 76.1% 23.9% BalanCD CHO Feedl l i in) Cell Vento Ciro-210 + Cell Verne 72.2% 27.8% (7) ActiCllO P Feed A, Feed B 69.9% i 30.1% l {@9292} In summary, AotiCHO P media is significantly better than the other media tested in regards to cell density, Viability and protein titer. onietry analysis indicates that the CD FortiCHO medium + CD Efficient Feed C has the highest percent of most active niultiniers {Table , hand 5+) at 35. 7%. However, the CD FortiCHO medium + CD Efficient Feed C has the highest percent of high molecular weight material that does not move into the gel at 4.7% (as seen at the top of the gel in FlG. l ‘7), suggesting that this media also generates a greater fraction of ated, un-ord ered niultiniers with fewer highly ordered and more functional ntultinrers than ActiCHO P.

ActiCHO P plus feed A and B has the second highest percent of higher order niultimer bands ahoye hand 4 at 30.l% with a negligible amount of non~specifically aggregated high molecular weight material. that does not move into the gel. Thus, -IO P likely has the t percent of fully functional higher order multimer hands. These data fuither indicate, surprisingly, that upstream manufacturing conditions not only affect the cell ity, density, and total protein titer, but also the tion of clinically efficacious higher order ers of (EL—2045. As demonstrated in Example 3 the highest molecular weight fractions exhibited decreased binding to FcRyllla, suggesting that the highest lar weight ons (6g. unordered GL~2045 aggregates) are less biologically active than highly—ordered inultimer of GLnZOAtS. As shown herein, very surprisingly, of all the basal media tested. only ActiCHO—P demonstrated high total Gl,~201l-5 protein titer, high, multimerization, as well as minimal amounts of unordered, high lar weight 61.,»2045 aggregates.

Altered Feeding Protocols {@8293} After ining that ActiCl—lO P media + feed resulted in optimal protein titer and production of higher order niultimers, altered feeding schedules were tested to determine whether one could attain similar or optimized results by g every other day~ As demonstrated in HS. l8, feeding every other day (blue) did not ly effect cell density (FIG. lSA), cell ation] yzus [Annotation] yzus viability (FIG. l8B), culture pl-l (FIG. l8C), or protein titer {Flt}. lSD) as compared to g every day. These results surprisingly te that similar results can be obtained with feeding every other day, and may be preferable for maintaining sterility and zing manufacturing costs However, similar experiments g every third day suggested that viability and protein tion may start to decrease with feeding less frequently than every 2 days (). {90294} Additional experiments were med to determine if ActiCHO l) Feeds could he used with other nonnActiCHO basal media to achieve similar results. Briefly, GL~2045 clone 58 was cultured in Power CEO-"l CD (Lonza, it 12m771Q) + 4-1an aniine (Lonza, # l7—605E) + 1X H'l Supplement (Gihco, it 11067—63 0). After passaging, the cells were washed and inoculated at 0.3 x'lt)6 cells/ml in AetiCHG P complete media AetiCHO l) + oniM LnGlutamine (Lonza, # 17- 605E) or Power CEO—2 CD complete media and cultured without temperature shift. ActiCHO P PowerCHO cultures were fed every day with + l niL ActiCHO Feed—A (FAA, #- Ul 5—072) + 0. l niL Feed—B (FAA, # [MS—0'34). On day 9, cell viability and protein titer were determined as described in Example 5 throughout the culture {Flt}. '30). The results show that, when used with ActiCHO Feeds A and B pursuant to the manufacturer’s recommendation, both PowerCHOZ and ActiCHO P generate the same cell viability and 045 protein yield.“ Thus, if multimer composition remains unchanged relative to ActiCHO P + A, + B ActiCHO Feeds A and B may be able to be used with other select highnperforrning base media.

Exam .le 7 »~ Otimal timin- and extent of tern-eratnre shift. for GL~2945 5 reduction } The e of this experiment was to assess the optimal timing and extent of ature shift. Numerous investigators have considered the effects of temperature shift on the cell cycle, apoptosis, and n'ietaholisin of a recombinant Chinese hamster ovary (CHO) cell line.

However, while consideration is generally given to the effect of temperature shift on the viable cell density, little if any attention has been paid to the required minimal cell density for optimal temperature shift results. Further, the minimal cell density is necessary for successfully conducting a temperature shift in multimerizing stradomers has not been ered. {99266} The present investigators surprisingly found that for optimal GLuZOllS expression and maximum titer, a minimum viahle cell density of 10 million cells/mL at the time of temperature shift is required. r, this requirement is more important than the day of culture at which the temperature shift takes place. The timing of the temperature shift is surprisingly most ation] yzus [Annotation] yzus successful when Viable cell density is in a logarithmic growth phase, generally when the Viable cell density is l0 — 15 million niL. This generally corresponds to day 4 5 of 'bioreactor cuiture depending on initial seeding density. {@6297} Furthermore, in a departure from what is described in the art, a temperature shift from 37 degrees centigrade (37o C) +/~ 1.0 C to 32.5C +"— 1.0 C is preferable to a temperature shift to 31° C -+—/— 0.5 C for optimal upstream conditions for manufacturing highutiters of (3L_2045, ’l“wo separate pools of Gl.,-2045 supernatant were generated from stable CHO clones using identical ions except for the nature of the temperature shift. CHO cells were cultured in a. l0L XER— lO Single—Use Bioreactor System ctor (Xcellerex, GE) with a pH shift from 7.1 to 7.0 at day . ActiCHO—P CD (cattt U21~GS l) 7 liters was used for production along with 280 ml daily ofPAA Feed A (cattt 3) and 28 mi daily of PAA Feed B (can't U l 5-054). PAA Feed A and B are equivalent to ActiCi-{O Feed A and Feed B. Temperature shift to 32. 5 degrees (A) and temperature shift to 31.0 degrees (B) each occurred on day 5. Results are shown in Table l l.

Table 11: Effects of ature shift on CHO coil viability and GLZMS yield )A—wrifierl yield Ear 21 ity Bay 22 viability Peak cell densit' .0 x 106 cells/niL (day 9) £9.13 x 106 ’mL Exam le 8 — Protein A column urification of (11144945 re uires more fre uent CIP procedures {W208} Gin-2045 was purified with by affinity chromatography (AC) with a protein A HiTrap MabSelect SuRe column (GEM l-0034—95) with a g buffer of 20 mM sodium phosphate, 0.15 M NaCl, pl-{ 7. 2 and was eluted with 0.1 M glycine, pH 2.7, ified GL—2045 was stored in 1X PBS, pH 7.0 (Quality ical, Inc. #1 19~069~l Ol ). AC (3132045 purifications were processed without colurnn Cleaning in Place (CW) procedures at the end of each run, CIP procedures typically involve flowing diluted sodium hydroxide (0.1 0.5 M NaOI-l) through the coiunin between purification cycles to hydrolyze deposits while sanitizing the protein A resin, thus regenerating the binding capacity of the protein A column (BouletuAudet er oi, Scientific Reports, Vol. 6, 2016). (EL-“045 was purified on 4 separate protein A affinity columns {columns 1—4, run 11.27.12}. The same protein A ty columns were then used for a second ation] yzus [Annotation] yzus affinity purification run (run ll.28.l2} after eluting the column with l00'33/ia elution buffer.

Purification of GLZO45 twice using the same column showed reduced amount of the lower lar weight species, both the homodimer and the dimer of the homodiiner, after a second purification run (), As shown in Table 12, densitometry analysis of SDS—PAGE demonstrated significant loss of honiodimer and dimer hands, indicating that the lower lar weight hands are outcompeted in binding to the protein A affinity columns by residual, highly protein A—avid, GLZCMS protein that is not completely d from the protein A column by elution with elution huffer.

Table 12: Densitoinetry of protein lled GL—204S Humodimer Band 1 Run 11.27.32 % Bifference Run 1/2 Column 1 Column 2 16.5 l Column 3 21.8 45.1 Column 4 22.2 mil ________________________________________________________________________________{arenas_______________ __________________________________________ Dimer Run 11,2112 Run 11.28012 Percent Difference Run Band 2 Column 4 {00209} Loss of lower molecular weight bands indicates that there is an avidity—based binding to the affinity column whereby high molecular weight rnultimeric GL2045 with multiple protein A binding sites outcompetes the low molecular weight species, causing a loss of the lower molecular weight species and effectively changing the composition ofthe drug, These data suggest that more frequent Cll’ procedures, and thus more frequent regeneration of the protein A column, are necessary for optimal purification of Cris—2045 when using protein A columns for multiple purification runs. These results were unexpected since, as bed above, regeneration typically requires the use of NaOH that would degrade the protein A columns most commonly used in the art. More nt use of such a buffer would thus result in faster degradation of the protein A column. As such, a protein A column capable of anding frequent CIP procedures with a trength NaOH buffer, such, as 0.5 M NaOH, must he used for regeneration of protein A [Annotation] yzus [Annotation] yzus columns used repetitively in the purification of (EL-2045 in order to maintain the optimal profile of the intact (EL—2045 drug.

Exam le 9C1? Procedures for re eneratino‘ rotein A for re etitive cation cycles of Gil—2045 rcgnires 0.5 M NaGH {@0219} Loss of low molecular weight species in the absence of Cll) procedures, as shown in e 8, also indicates that there is high molecular weight species that remains bound to the protein A affinity column after the first column run, thus occupying the g sites and ting the binding of lower molecular weight s in subsequent purification runs. These data indicated that additional Cl? procedures should he employed to maintain the multimer profile of protein fied Gila—2045. {(39211} The t inventors ered by r, daily cation using the HiTrap MahSelect column (GE #28—4082—58) that after approximately 6 — 7 purification runs, the composition of the purified (EL—2045 changed, marked by a subtle loss of the homodimer and dimer fractions. A skilled artisan purifying, for example, a monoclonal antibody would not expect to find a change in the composition of the purified t after 6 — 7 protein A purification runs.

To solve this problem, Cl}? procedures with the manufacturer’s recommended 0. l M NaOH were performed to regenerate the binding capacity of the protein A. column, These CIP ures were performed after each purification run, which is more frequently than used in the art. Employing frequent (ZIP procedures resulted in some improvement, but did not resolve the problem ol’a. loss of the lower molecule weight species. The inventors thus deduced that the avid binding of (31.4“ 2045 to n A requires a more stringent (ZIP regimen than a skilled artisan would normally use in order to fully regenerate the cohimn in order to facilitate retention of the hornodimer and dimer. {@9212} However, the resins commonly used in n A columns (cg, MahSelect} are not NaOl-l resistant and would therefore quickly degrade with the use ol‘a more stringent NatH-l buffer, and such degeneration is associated with diminished purification capacity and with a change in the Gals—2045 multimer composition. .l-lowe‘ver, less commonly used protein A media tag, elect SuRe (General Electric #l 1—0034—95)), can withstand enhanced cleaning at 0.5 NaOl-l. As such, the inventors used a MahSelect SuRe column with daily (Ill? procedures using a 0.5 M NaOl-l buffer. After the implementation of more frequent and more stringent Cll’ procedures, protein A [Annotation] yzus [Annotation] yzus purification of 45 was accomplished on a daily basis without loss of homodinier or dimer and without change in the ition of GLuZOZlS. {@9213} Thus, the ors discovered that protein A column (Ill) of GL—ZOL’lS es more stringent and more frequent Cl? procedures than would normally he employed by a skilled artisan working with a monoclonal antibody or Fc fusion protein in order to retain the honiodimer and dimer, and thus the optimal profile, of the intact GLnZOZlS drug.

Example 10 ~— l’rotein A column 32H elution gradients are used to purify GL—2045 of heinodimer aggregates that are not highly ordered multimers {902114} pH elution gradients are commonly used with protein A columns during n purification to optimize for total protein yield; but are not typically used to change the composition of a drug. The present inventors discovered that such pH n gradient on a protein A column can be used to eliminate unordered aggregates of GL—ZOZl-S from the higher order mers. GL— 2045 CEO supernatant was purified by affinity chromatography (AC) with protein A HiTrap MahSelect SuRe (Glittl l~0034m95) with a binding buffer of 20 mM sodium phosphate, 0.15 M NaCl pH 7.2, followed by an additional. wash. with. the binding . (EL—2045 bound to the protein A was eluted by an elution buffer comprising 0% to l00% of 0.1 M glycine pH :27, thereby creating a pH gradient for (113045 elution from the protein A affinity column. Eluate fractions were collected into a 96 well plate er bio-one #780271), and neutralized at plrl 7. 5 by adding Tris-l-lCl pH 9.0 into each well. lent protein amounts of each of the fractions were then run on a non~reducing SDS~PAGE gel (Ll—12% NiiPage Bis~Tris, lnvitrogen at NPO3ZZBQX). {90215} AGE analysis of the fractions obtained by n of the protein A column with a pl-l elution gradient demonstrated that very high molecular weight species were eluted last in on D6 and D7 (FlG. 23A and Flt}. 23B). Surprisingly, the very first fraction (Di) also contained high molecular weight species. As such, these results indicate that high molecular weight fractions can he separated from the main species (cg, honiodimers, dimers and higher order multimers) of GL-ZO45 by pH gradient elution of n A. affinity column. As shown in Examples l—3, these fractions may represent lower activity s demonstrated by diminished Fc receptor binding and diminished CDC inhibition. {99216} Also shown on gel (MG. 238, far right lane) is a very high molecular weight fraction obtained by regeneration (Cl? by 0.5M NaOH and neutralized by HCl). These results [Annotation] yzus [Annotation] yzus indicate that there is high molecular weight species residing on the protein A column after elution indicating again a need for high stringent NaOl—l buffer during Cll’ procedures to regenerate full column g capacity. {@6217} 'l7heret‘ore, though not normally used for this purposed, pll elution gradients can he used to remove separate the high molecular weight, unordered aggregates of Gig—2945 from the ically active lower and higher order multimers. Such tion may be employed to further ze or maintain the multimer profile of ed 45. Alternatively, a step elution gradient may be used to arrive at an optimized GLn2045 composition.

Example ll m lon exchange column salt and pH conditions modifz GL—Z'MS multimer composition } An important goal for purification of Gl_,-2045 is the tight control of the niultimer composition of the final, purified product. ion exchange columns (cg, cation and anion exchange) are routinely used for polishing steps of purification of monoclonal antibodies and Fe fusion proteins. The present ors tested the cation exchange medium, POROS ClEX (lnvitrogen GoPure XS (10 ml.) eat it 4448885) with elation buffers comprising different concentrations of salt. Gl_.—2045 was first purified by protein A affinity chromatography, and then pooled and dialyzed in. 50 mM Sodium Acetate, pH 5.0 prior to loading on the CIEX column. 50 niM. Sodium Acetate, pH 5, was used as equilibration and wash buffer. The effects of elution buffer (EB) salt content on Gl..—2045 polishing were tested with a 50 mM Sodium Acetate elution buffer with varying amounts of buffer B added (lM NaCl, pl-l 5, shown as % in FlGr 24). Chromatography runs were performed on AktaAvant. Briefly, the Avant method comprises brating the CIEX column with 50 mM Sodium Acetate pl-l 5 at 2 i'nl,/’rriin at a total volume of it) column volumes (cv). 65 to 100 mg of GLu2045 that was previously dialyzed in 50 mM sodium acetate, pl-l 5 was loaded onto the column. The column was washed with 5 cv of binding buffer at 2 mL/min. GL- 2045 was then eluted with between 9 and ii cv of buffers comprising varied percentages of NaCl (cg elution s comprised of 306094) buffer B) at 2 mL/min . {89219} The 9/1) recovery of GLuZOL'lS was determined for GL~2045 eluted with elution s comprising 39% buffer h, 40% buffer h and 50% buffer b (39% EB, 40% EB and 50% EB, tively) to determine the optimal range of salt concentrations for elution buffers (MG. 24).

[Annotation] yzus [Annotation] yzus These data demonstrated that alterations of the salt content in the elution buffer can substantially modify the multiiner composition of {Ho—2045.

Table 13: % reeovery of GL—2045 eluted with elation s comprising 30=50% buffer h Elation Ste % of load ——SIM-5A3' 3.0 -—531—513340 6A1—6A3 4 l {86220} As shown in Example ll and quantitated in Table l3, SEES—PAGE analysis demonstrated dramatic differences in separation of Gl_,-2045 molecular weight s when eluted with 30% EB, 40% EB, or 50% EB elution buffer. 9i 7% of the n'iaterial was ehtted with an n buffer comprising 30~40% buffer h, and the material recovered with an elution buffer comprising 50% buffer B was only high molecular weight material. These data demonstrate that alterations of the elution buffer can substantially modify the imiltirner composition of 45, Thus, it is clear that the elution buffer selected for the ion exchange can he used to modify the innltirner composition of (EL-2045, a novel use of this technology, At the same time, the inventors have discovered that, if no change in composition of (EL—2045 is desired in the ion ge polishing step, a level of precision not normally practiced by the skilled artisan is required in ing the salt concentration of the elution buffer for use in the ion exchange polishing steps. {90221} As 9l.7% of the {EL—2045 material in Example ll was eluted with elation s sing n 30-49% buffer h, and the material recovered with elation buffers comprising 50% buffer B was only the high molecular weight material, elation buffers with a buffer ‘0 range of 30—4 % were ed for further analysis. The methods employed were similar to those described in Example ll. {$9222} The SDSJPAGE analysis demonstrated dramatic differences in separation of GL— 2045 hoinodimer when eluted with elution buffers comprising 35%, 36%, or 37% or higher of buffer h (3 5% EB, 36% EB 37% EB, etc, res ectively‘ . Note that the homodimer and dimer of t l3 - [Annotation] yzus [Annotation] yzus the homodiiner that are clearly Visible at 35% EB are, in contrast, y d at 36% EB and tely eliminated at 37% EB and higher (FlG. 25), Similarly, the SDS—PAGE analysis demonstrates dramatic differences in separation of the highest order inultirners of (EL—2045 and large unordered aggregates when eluted at 35% EB 36% EB, or 37% EB, or when eluted with elution buffers with higher buffer ‘0 concentrations. Thus, it is clear that the elution buffer selected for the ion exchange can he used to modify the multimer profile of GLn20457 a novel use of this logy. It is also apparent that the data from this step elution can he used by a skilled, artisan to select a single step or multiple step elution to ohtain the desired GL—2045 profile. For example, GL—2045 polishing using a POROS CIEX column and an elution buffer comprising 36.538596 buffer ‘0 will retain all homodimer, dimer, and, multimers h multinier 10 and will elute off the large unordered aggregates and the t order multimers. A similar example using a different column is shown in e 4.

Examgle 13 m ion exchange chromatin?raghy can he used to reduce or eliminate large hoinodimei' aggregates and the highest order mnltimers {(30223} Mannfecturing of optimal itions of 045 require minimizing the amount of the highest order {EL-2045 multimer (erg hands above the clearly delineahle hand l0, imately 600K?) and eliminating material above lOOO kl) to remove both large homodimer aggregates and the t order rnultimers whose increased valency confers increased theoretical risk of inunnnogenicity. (EL-2045 was eluted with elution buffers comprising different percentage ofhnffer h (38951323 (Cl )7 39%EB ((12 and 40%EB ( 3. 26). Main elution peaks for 814'“ , 13)) (El 2045 were observed at 38% (Cl ), 39% (C2), and 40% ((33) {FM}. 2.6), followed by smaller elu‘tion peaks at 50% and 100% buffer B. The % of (EL—2045 recovery for each elution buffer was determined and is represented in Table l4.

Table 14: Percent Yield of (Sllu2045 with varied elation buffers % Buffer B % Yield % Buffer B % Yield [Annotation] yzus [Annotation] yzus Waste 24} Multimer profiles of the eluted GL-ZOL’lS fractions were ined by visual inspection of SDS~PAGE analysis (FK1 2?). Visual inspection ot the SDSPAGE analysis of eluted fractions ted that 38/o of elution buffer red G1@045 with the least amount ot residual l'iigh lar weight material. {00225} {EL-2045 peaks were quantified by densitoirietiy (HQ. 28, summarized in Table l5 as percent intensity in SIDS-PAGE hands). l’eak ll represents the homodimer with the lowest molecular weight and peak 1 represents the material with the highest molecular weight material that is preferably eliminated.

Table 15: Summary ol‘Densitometric Analysis AC/CEEX 39% AC/CIEX 40% AC {@3226} These results demonstrate that a step elation protocol with an acetate elutiori buffer sii'ig 38% or 39% butler B at pH 5 yielded approximately 85% of the preferred fractions of 611-2045 and reduced the higher molecular weight fraction above 600er. Although visual inspection of S.DS~PAGE indicated that the high molecular weight material was lowest in the [Annotation] yzus ation] yzus fraction eluted an elution buffer comprising 38% buffer h, densitonietrio analysis indicated the lowest percentage of the highest molecular weight fraction was obtained with an elation huffer comprising 39% buffer ‘0. However, densitometrie analysis demonstrated that all of the ClEX purified protein compositions ned smaller amounts in high molecular weight on (hand 1), compared to material purified by affinity chromatography alone. l, analysis of the eluate suggests that the amount of large aggregates and highest order multimers can be controlled by applying controlled elution conditions with the Pores ClEX column. Thus, it is clear that the elution buffer ed for the ion exchange can be used to modify the large aggregate and largest highly ordered multinier ition of GL—“(Mi a novel use of this technology A similar example using a different column is in Example 4.

Exam le 14 m Modification of GLZMS multimer com osition with hydro liohie interaction chromatography columns {(39227} 45 was produced from a stable HEK 293F cell line and was grown in 293 Freestyle Media o #l2338—Ol8) with Glutamax (Giheo #- 35050—06l) and Genetiein (Gibeo #10131—027). Supernatants were harvested twice a week and were filtrated at OZum into ll, filter system, (Corning #43l093). (EL-2045 supernatant was then ed with protein A HiTrap MabSeleet SuRe (GEM 10034—95) with. a binding buffer of 20mh/l’. sodium phosphate, 0.15M NaCi, pH 7.2 and eluted with (ll M sodium citrate elution buffer with a pH of 3.0 36. AG purified 45 was stored in lX PBS pH 7.0 (Quality Biological, lne. #l 19—0694 Oi). {(38228} GL-ZO45 was then purified on 7 different l-lydropliobio interaction Colunii'is (HIC) using l-liTrap HlC Selection Kit (GE; #28-4l lO-07), Columns included in this kit are described in Table 16.

Table '16: l—lydrophohie Interaction Columns Columns CV Hi’l'ra But 'l HP __ ___t:t_i_ilfrari__51?;i3areitfi_tl_eu_§atai_____ Hi’l‘ra Butyl FF l-li'l‘rap ButylmS FF HiTra 3 Phenyl FF high Sula 7 Hi ”l”rap Phenyl FF 1 niL [Annotation] yzus [Annotation] yzus {00229} El] C columns were brated with 50 mM Sodium Phosphate, l.0 M ammonium sulfate, pl-l 7.0 (Start Buffet) and 3.5 mg of AC-purified lS (diluted in 4 volumes of start buffer) was loaded into each column. After washing with the start , a gradient elution was performed using 0% to 100% 50 mM Sodium Phosphate, pH 7.0. All onated peaks and flow through were tested by SDS page. The non—reduced samples were loaded into 15% Tris ElCl (Brion Radiil til-l. 15) The staining was done using Silver Stain kit form lnvitrogen #LCo'l 00. 309230} The seven different Hi (I s demonstrated different multimer profiles of Gb 2045. As an example, the butyl HP column separated the homodimer fraction (fraction AlO) from the eric molecular weight species found in AlZ. The same effect was seen with the phenyl HP column (fraction Bil compared to fraction BS). Only loo/E; of loaded al was recovered in the elution fractions with the Qctyl FF column, indicating that it may he well suited for a flown through mode polishing method for GLnZO45. Further, the eluted fractions from the Octyl EF column contained the higher molecular weight species, indicating that the column may also be well suited for removal of very high lar weight homodimer aggregate species that have lower potency. {09231} A, similar experiment was performed on the marine version of (EL—2045, known as M045, M045 was purified by n A affinity chromatography and then further purified on the AKTA Avant (GE) by BIC with Hiload 26/10 Phenyl Sepharose High Performance (GE l 7~l086~ Ol). The BIC column was equilibrated with (ll M sodium phosphate, l M ammonium sulfate pH 7.0 (Start buffer) and M045 was loaded onto the column followed by a wash step with the Start buffer to remove all unbound materials. M045 was then eluted with {ll M sodium phosphate pH 7.0 eluti on buffer with a, gradient elution (—3 l ). {(36232} EEC-purified fractions of M045 were analyzed by SUE-PAGE to determine the effect of the EEC column polishing on the MO45 multimer profile. These results further demonstrated that hydrophobic interaction columns can he used to modify the multimer composition ofM045, noted by the clear separation of the homodirn er, dimer, trinier, and niultinier fractions (F1G. 32}.

Exam le 15 — Exam law 5 rotoeol for otimallz reduced (E'EJuZMS ation] yzus [Annotation] yzus {89233} The data described herein trate the optimal conditions for several variables of the upstream and ream manufacturing process for (EL-2045 that result in optimization of (1) protein titer, (2) cell viability throughout culture, and (3) multimerization ot‘ (EL—2045 and (:4) maintenance of the m ultimer profile in the final GL~2045 drug nce. lniportantly, the level of multimerization of (EL-2045 is critical to the clinical efficacy of the stradomer (See Examples ln 4). Current culture methods are not necessarily aimed at the optimized production of a specific fraction or enhancement of a particular erization pattern. As such, the upstream culture reagents and conditions and the downstream purification media and conditions that affect multinierization are all n and cannot be predicted based on the current state of the art. {@0234} The data described herein ed in the discovery of the following protocol elements for generating optimally ctured {EL—26,345: {99235} ’ , The o ~itiimal based media. for Generating o timall ' manufactured (EL—2045 is ActiCHO P, {@9236} Data described herein demonstrate that the optimal base media was ActiCHO P.

CHO cells cultured in a hioreactor in ActiCHO P base media resulted in a high cell density and cell viability, while optimizing, for an increase in protein titer ed to other base media tested.

Surprisingly, ActiCHO P media resulted in an increase in the tage ofhigher order ers of Gl.,~2045 present at the end of the culture protocol. {linear} ‘ The o itinial feeds for eneratin r o tirnall ‘ manufactured CELT/2.045 is ActiCl-lO P are ActiCl-lO Feed A and Feed B, {(38238} Data described herein further demonstrate that the optimal feed was ActiCl-lCE P Feed A and Feed B, added to the culture every day or every other day. ActiCl-lt.) 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 media/feed combinations tested. lmportantly and unexpectedly, ActiCllO P media with Feed A and Feed B ed in a high level of highly ordered multimers and, importantly, a reduction in the percentage ot‘highunolecular weight, unordered aggregates of GL~2045 compared to other media/feed combinations tested. These data indicate that this particular media/feed ation results in the production of a greater tage of GL—2045 multiiners with enhanced clinical efficacy (cg, a greater percentage of highly ordered (ESL-2.045 multimers). The present inventors surprisingly found similar s for addition of Feed A and [Annotation] yzus [Annotation] yzus Feed B every other day, indicating that this particular media/feed combination can he used to reduce costs and mitigate the rislt: of contamination associated with daily culture lation. {@9239} Further, —lO P media with Feed A and Feed B resulted in the production of a substantial percentage of GLu2045 ng as higher—order multimers, while minimizing the percentage of unnordered, high molecular weight aggregates of GL~2045. As such, this ular media/feed combination surprisingly optimized specifically for the biologically functional and ally efficacious fractions of highly ordered multimerized 45, therefore optimizing retention of the GL-2045 rnultimer e While reducing the need to eliminate high order aggregates in further downstream purification steps. {902149} The o tinial tem Jerature shift for >eneratin r o itimall manufactured GL~2045 is.ashi_tit__fi:oin_3__2_i__§___t__r_2__?2_Z_.__§_f__Q__l2ascrl_nn_izsl_l___c_i_erisitr_._ {99241} Data described herein additionally demonstrate that a temperature shift from 370 C to 32.50 C based on cell density results in optimal cell density, viability, and protein titer. This temperature shift protocol is a deviation from ished ols (Ouguchi at a], Cytotechnology, 52(3), pp. 199—201 (2006); Masterson and Smales, Pharmaceutical Bioprocessing, 2(1), pp. 49—61, (.20l4)), which describe a temperature shift from. 37° C to 31'3 C hased simply on day of culture. The present inventors unexpectedly found shifting the temperature to 32.5“” C after the cells had reached a density of r-le-l 5 x 105 mL resulted in not only the maintenance of a high cell density and cell viability, but also in a substantial increase in protein titer compared to previously established protocols. {99242} Data demonstrated herein indicate that specific downstream purification protocols result in GL~"045 itions with an optimized inultiinerization profile. In carrying out these purification methods, strict attention must he paid to maintaining the desired multimer profile of {EL—2045 by controlling column ions and buffers. This stands in stark contrast to a monoclonal antibody, Fc fusion protein, or similar CEO-derived protein where purity and retention of yield are the primary goals. } '4 O timized irotein A urification of 5 re uires fre uent and strinoent Cll’ methods. {$0244} GL—2€)45 avidly binds protein A. This avid, binding resulted in (EL—2945 remaining hound to the protein A media in the column when Cll) procedures normally employed for mAh purification were used. As a , with repeat cycles of use of the protein A column, the [Annotation] yzus [Annotation] yzus homodimer fractions of (EL—2045 were unable to bind to protein A and flowed through the column.

This resulted in a ntial and functionally important change in the rnultimer profile of the final protein tilt—purified (EL—2045 product. The avid binding of (lL—2045 therefore resulted in a requirement for more nt and more stringent (eg, using a 0.5 M NaOH wash buffer) (Ill-‘3 procedures than are commonly used in the art (egg during mAb or tic—fusion protein purification).

These results were unexpected. as most commonly used protein A columns are unable to and the stringent NaOH washes required to remove GL~2045 rnultimers and to fully regenerate the protein A column. ore, only some protein A columns, especially MahSeleet SuRe, are e of being used for GL-ZO45 cation and will require frequent CIP procedures with approximately 0.5 M NaOH to maintain the d GLnZt‘rdfi multimer profile. {@0245} 5 ' H elution gradient or ste . A elution facilitates the se aration of the hi ihest molecular weight fractions of Git—20:45. from the lower and hi” her order multiiners. {£30246} Using pH elution gradient with protein A purification resulted highest molecular weight components being eluted in the first and last elution fractions. These data indicate that a pH gradient can be utilized to te the ically active ons of 5 (tag, the homodimer, dimer, and higher order multimers) from fractions comprised of the un—ordered high molecular weight aggregates? which have been previously shown to have decreased biological actiVity. 7} 6‘ The o tirnal elution buffer for the )olishino of 81,4045 bl lon Exchan {(38248} lon exchange tography is commonly used to polish the drug of irnpurites during mAb production. However, the present inventors utilized ion exchange chromatography to eliminate specific ons of GL—ZOdS (eg, the highest order multimers and high molecular weight unordered aggregates of the homodinier) such that an optimal multirnerization profile was achieved. In ular, the present inventors found that an elution buffer of 30—40% buffer b decreased the amount of high molecular weight unordered aggregates of GLuZOZlS. Even more specifically, an elution buffer of 38—39% buffer b specifically maintained the amount of the hornodimer present in the final (EL-2.045 product, while also optimizing for reduced amounts of the unordered aggregates. {89249} ’- 7dro )hohic interaction columns :l-llC can he utilized to e s ecific (ll; .4045"l multimerization profiles [Annotation] yzus [Annotation] yzus {89256} Data herein demonstrate that multiple Hle can be used in the polishing steps of purifying GLQOL’lS. For example, flow through from the Qctyl FF column contained mainly high molecular weight species, indicating that this column can be used specifically for the removal of high lar weight aggregates of (EL—2045. Alternatively, the butyl l-l'l?’ columns can be used to te the homodimer fraction from the niultimeric fractions for application wherein one of the fractions may achieve more desirable outcomes. Alternatively, HlC columns can be used in g mode.

Optimized Alfai‘ztgfacturmg Protocolfirr (HZ—2045 {(130251} Taken together, incorporating all of the parameters discussed above, the following protocol resulted in the highest n yield of (EL-2045 while ining the highest percentage of the overall population as multimers. {89252} Cl-lO cells were transfected with two vectors using a proprietary transt‘ection system by Gene SA (Monthey, Switzerland), one a 5 expression vector comprising a GL~ZG45 expression te flanked by piggyBac osase targeting sequences, and the second vector comprising a piggyBac transpoase. PiggyBac transposon has preferential insertion into highly transcribed regions of the genome and additionally contains inverted, terminal repeats that provide insulation from gene silencing The transfection resulted in the integration of the expression cassette into highly ribed genomic regions thereby establishing a bank of stably transfected CHO cells with fewer than 20 genome insertions of the transgene. The stably transfected CHO cells were then. cultured in a bioreactor with ActiCHO P media at a growth temperature of 37° C, During this culture, cells were fed daily with ActiCHO Feed A and Feed B at a growth. ature of 37° C, until the es reach a cell density of about 10 million to about n cells/inL After such densities were reached, the growth temperature was d from 37° C i 1°C to 325°C i 1°C, and optimally manufactured GL—2045 from culture media was harvested from the media on the final day of culture. {89253} This protocol resulted in a cell viability of greater than 95% at day l8 of culture and greater than 80% at day 21, and a final total protein titer of greater than 9,000 ing/mL) wherein greater than 70% of GLu2045 was present as nonuhoinodiniers and greater than 30% was present as higher—order multiniers above the fifth rnultinrer.

[Annotation] yzus [Annotation] yzus {89254} GL—ZO-tS was harvested from the culture supernatant with a tangential flow filtration system that does not obstruct passage of the largest highly ordered rnultirners, thereby retaining the homodimer and multirner profile of the supernatant. Downstream manufacturing methods were then employed to isolate (EL—2045, to remove impurities, and to e a particular fraction in order to control the rnultimer profile of GL—2045 (cg, removal of tin—ordered, high molecular weight aggregates). {EL—2045 was purified, by protein A affinity chromatography, wherein protein A media was ed for the ability to withstand high nity ration.

Further, more than one wash huffer was used to enable further control over the cation process.

Additionally, Cl]? procedures were med more frequently than normally done and with a 0.5 M NaOll buffer to remove GL—2045 multimers that had avidly bound to the column in order to fully regenerate the binding capacity of the protein A column as required to retain the honiodirner in the final Glut—2045 composition, GL~2045 was eluted from the protein A column with or without a pH elution gradient, After purification by protein A column affinity chromatography, additional ing steps were ed. Cation exchange chromatography was used to remove high molecular weight, unordered aggregates of (EL—2045 with an n buffer comprising 37—39% +/— 0.5% buffer b, preferably with a CIEX FORD-S XS resin. In some embodiments, HIC s were used to further purify (EL-2045, To remove high molecular weight, red aggregates of GLQO45, the Octyl FF resin. was used as an additional polishing step. Alternatively, the hutyl H11 containing columns were used to isolate specific GL—ZMS tractions (cg, isolation of the higher order inultimers). Additionally, anion exchange columns, specifically Q Sepharose Fast Flow columns were used as an additional polishing step, particularly in flow through mode. {90255} While these additional purification steps may he used individually, it was preferable that purification of (IE—2045 by protein A affinity chromatography was used in combination with all three of anion exchange, cation exchange, and hydrophobic ction tography to arrive at a final (Ills—2045 drug substance, wherein the final protein titer is > 4 g/,L of which ia of {its—2045 is present as a multimer, wherein >30% of the multimers are pentarneric multiniers or higher.

Exam le 16 Analysis ofo .timallv reduced (lb-2045 {89256} To further characterize the optimally ed GL-ZOdS made by the methods described herein, GL—ZO45 was produced in a hioreactor according to the am methods [Annotation] yzus [Annotation] yzus described herein using the ActiPRG basal media, feeds, temperature shifts described herein. The ing (b20453 supernatant was then passed through a Millipore XOHC depth filter followed by filtration through a 0.2 [.1131 filter and processed using multiple downstream processing methods.

The multimeriza‘tion profile of the GL2045 composition was assessed after each processing step and is shown in F1 G. 33. The multinieriza‘tion profile of the filtered (EL-2045 preparation is shown by the red dots in . The filtered (EL—2045 preparation was then ted to affinity chromatography with protein A MahSeleet Sure (GE. till-003495) (multimerization profile shown by blue dots in }, and then ed with AlEX in flow through rnode using the Q Sepharose Fast Flow column. The ing SDI/1045 was then pH adjusted to pH 5.9 :t 0.10 and filtered through a 0.2 pm filter (rnultinrerization profile shown by green dots in F1G. 33). The GL— 2045 was then purified with cation exchange chromatography using the l’oros XS column in binding mode by step el'ution using an elution buffer comprising 50 mM NaAcetate + 375 mM NaCl, pl-l 5.9 (rnultirnerization profile shown by yellow dots in HQ. 33) before hobic interaction chromatography (multimerization profile shown by orange dots in Fch 33), and filtration (niultinierization profile shown by purple dots in ) to arrive at the final drug substance (multimerization e shown by black dots Fig 33). Raw data for FIG 33 is shown in Table 17 helow.

Table 17: Multimer' pereentages determined by analytical EthLC Hl-l RegTox lMabi elect Run 1 Load HH RegTox :2 MabSelect ___l:{_.1__u_i___1 Elli Reglon 3AEX Pool Run 1 till Reglox 4 CEX p00} Run 1 HH RegTOx HlC Pool Run 1 HE RegTOX <3 UFDF Pool Run 1 HH RegTox ____R_u_r_i __l iii-iRégro'x'" ' ll‘vltbSclect Run4 Load ’ Hl-l ox 2 MIahSelect Run 2 Pool ation] yzus [Annotation] yzus “1”“?I“ 3 AEX Pooi Run 2 RH Reg I0); 4 CEX P001 Run 2 HH Reg“ HIC Pool Run 2 HH RegTox i HE Reg’i‘a Run 2 HH IDOL Run 1 HH IQOL Run 1 HE 100}; 3 AEX Pool Run 1 HH IOOL CEX P001 HE EOOL 1-110 P001 Run 3 HH EGOL 6 UFDF Poo}.

Run 3 HH 'EOOL Run 1 HH EGOL 1MabSelect Run 2 Load 2 MabSelect , . : AEX P00}, 4 CEX P00} Run 2 HH IDOL HEC P001 Run 2 HH EOOL <3 UFDF P001 Run 2 HH IOOL Run 2 HH lOOL Mastlect Run 3 Load HH EGOL ‘2. MabSelect Run 3 P00} HH 'EOOL 3 AEX Pool Run 3 HH 'EGOL 4 CEX P001 Run 3 ‘ HIC Pool 6 UFDF Pool Run 3 HH EBOL Run 3 HH 25014 1MabSeleci Run Load ation] yzus [Annotation] yzus RH 250i. 2 MabSeleet Run P001 .VH“ AOL._ . , q E 3 AEX Poet Run a HH ZSOL 4 CEX P001 BIC Pool UFDF Pool eie<rt n GMP i Load 2 MabSelect 53 GM? 1 Pool JJ GM? 1 5 001 t 4,. 33 GIMP} SHIC P00} 31.80 ' > JJGMP i UFDFPOOE .r _ .

JJGMP 1 es lMabSeiect JJ GMP 2 Load QMabSelect n GMP 2 P001 new xtexnijooi“ , ____ _ ___, n GMP 2 cex Pool 33 GM}? 2 5 Hit: P00] 33 GM}? 2 UFDF Poe} ------------------------------------ , J, Us ' , , J3 GM]? 2 1 MabSelect H GMP 3 soad 2 MabSelect JJ GMP 3 Pool 33 GM? 3 AEX Poor 13' GM? 3 Ciex Poo} JJ GM? 3 5 HIC P00] 33 Gm2 3 6 UFDF Poet {953257} The percentages of the homodimer, dimer, trimer, tetramer, pentamer, hexamer and 7+mer fractions were assessed after each step by anaiytieal HPLC. Briefly, supernatant from GL 2045 stably transfected CHO was generated ing, te the upstream methods described herein.

GL—ZO45 was next purified awarding tie the downstream methods described . Samples were obtained at the following successive stages of purification: Pretein A MabSeiect SuRe lead, [Annotation] yzus [Annotation] yzus Protein A MahSelect Sulte pool, Anion exchange pool, Cation exchange pool, HIC pool, UFDF pool, and Drug Substance. The s were compared by analytical SEC—l-{PLC Briefly, tic separation was performed by l-lPLC using two SEC columns nt Bio SEC (300143)) in series with UV detection at 280 nm on a High mance Liquid tography System (Agilent llt‘iO HPLC system). tography is performed with a run time of 60 s, and a flow rate of 0.5 mL/min. The relative area percent of each peak is calculated. 3130258} The results are shown in As apparent in FIG. ’33, the downstream processing of the GL—Ztlélfi altered the levels of the smallest fractions, the honiodimer and the dimer of the homodimer, as well as the largest fraction, the 7—mer + while fractions 3—6. remained quite . With respect to the smaller multimers, the progressive downstream manufacturing steps, from the protein A column loading through to the final drug substance, resulted in an increased recovery of the homodinier and the dimer of the honiodimer. However, the progressive downstream processing steps had the opposite effect on the highest order multimers resulted in a decrease in their relative percentages. {(39259} The resulting (EL—2045 drug product had a defined multinier pattern which comprised, as a percentage of the total composition, less than about 20% homodimer, and more than about 28% of the 7—mer and above. The composition also comprised about 7-1 % dimers of the homodimen about 6~1l% triniers of the homodinier, about l. (246% of the tetramer of the hornodirn er, about 63% of the pentamer of the dirner, and about MM 4% of the hexamer of the hornodimer.

Claims (36)

1. A composition comprising a recombinantly produced 5 homodimer, wherein the homodimer comprises less than 20% of the total ition.

2. A composition sing a recombinantly produced GL-2045 homodimer and a recombinantly produced GL-2045 multimer, wherein the GL-2045 homodimer comprises less than about 20% of the total composition, and n the GL-2045 multimer comprises: (a) a heptamer of the homodimer and above comprising at least about 28% of the total composition; (b) a dimer of the homodimer comprising from about 7% to about 13% of the total composition; (c) a trimer of the homodimer comprising from about 5.5% to about 11% of the total composition; (d) a tetramer of the homodimer comprising from about 10% to about 16% of the total composition; (e) a pentamer of the homodimer comprising from about 6% to about 10% of the total ition; (f) a hexamer of the homodimer sing from about 10% to about 14% of the total composition; (g) a dimer of the homodimer through a hexamer of the homodimer comprising from about 39% to about 61% of the total composition; (h) a trimer of the homodimer through a hexamer of the homodimer comprising from about 32% to about 50% of the total composition; (i) a tetramer of the homodimer through a hexamer of the homodimer comprising from about 26% to about 39% of the total composition; (j) a pentamer of the homodimer through a hexamer of the homodimer comprising from about 16% to about 23% of the total composition; or (k) any combination of (a)-(j).

3. A composition comprising a recombinantly produced GL-2045 homodimer and a recombinantly produced GL-2045 er, wherein the GL-2045 homodimer comprises less than about 20% of the total composition, and wherein the GL-2045 multimer comprises: (a) a heptamer of the mer and above comprising at least about 28% of the total composition; (b) a dimer of the homodimer comprising from about 7% to about 13% of the total composition; (c) a trimer of the homodimer comprising from about 5.5% to about 11% of the total ition; (d) a tetramer of the homodimer comprising from about 10% to about 16% of the total composition; (e) a pentamer of the homodimer comprising from about 6% to about 10% of the total composition; and (f) a hexamer of the homodimer comprising from about 10% to about 14% of the total composition.

4. A ition comprising a recombinantly produced GL-2045 homodimer and a recombinantly produced GL-2045 multimer, wherein the GL-2045 homodimer ses less than about 20% of the total composition, and wherein the GL-2045 multimer comprises: (a) a heptamer of the homodimer and above comprising at least about 28% of the total composition; (b) a dimer of the homodimer through a hexamer of the homodimer comprising from about 39% to about 61% of the total composition; (c) a trimer of the homodimer through a hexamer of the mer comprising from about 32% to about 50% of the total composition; (d) a tetramer of the homodimer through a hexamer of the homodimer comprising from about 26% to about 39% of the total ition; and (e) a pentamer of the homodimer through a hexamer of the mer comprising from about 16% to about 23% of the total ition.

5. Use of the composition of any one of claims 1-4 in the manufacture of a medicament for the treatment or prevention of an inflammatory, autoimmune, or infectious disease or disorder.

6. A method for producing a composition comprising a homodimer and/or a multimer of a mer, wherein the homodimer comprises two monomers each sing amino acids 21-264 of SEQ ID NO:4, the method comprising: (a) culturing Chinese Hamster Ovary (CHO) cells that have been stably transfected with an expression vector comprising a nucleic acid sequence encoding SEQ ID NO:4 at 37ºC ± 1ºC until the CHO cells reach a cell density of about 5 to about 30 million cells/mL; (b) shifting the growth temperature from 37ºC ± 1ºC to 32.5ºC ± 1ºC; and (c) ting the homodimer and/or the multimer of the homodimer from the culture media.

7. The method of claim 6, wherein the cells are grown to: (a) a density of about 10 to about 25 million mL prior to the shifting growth temperature; (b) a density of about 10 to about 15 million cells/mL prior to the shifting growth temperature; or (c) a density of about 15 to about 20 million cells/mL prior to the shifting growth temperature.

8. The method of claim 6 or claim 7, n the culturing is performed in ActiCHO P base culture media, or a base culture media with substantially the same constituents as ActiCHO P, and n the CHO cells are fed during culture with HyClone Cell Boost 7a and HyClone Cell Boost 7b.

9. The method of any one of claims 6-8, wherein the sion vector comprising the nucleic acid sequence encoding SEQ ID NO:4 comprises a nucleic acid sequence encoding the leader peptide of SEQ ID NO: 1.

10. The method of any one of claims 6-9, wherein the expression vector comprising a nucleic acid sequence encoding SEQ ID NO:4 further comprises a piggyBac transposase recognition sequence and is transfected with a vector encoding a piggyBac transposase.

11. The method of any one of claims 6-10, wherein the expression vector results in less than 20 c insertions.

12. A recombinantly produced composition comprising a homodimer and/or a multimer of the homodimer made by the method of any one of claims 6-11.

13. An expression vector comprising an expression cassette comprising a c acid encoding SEQ ID NO:4, wherein the expression cassette is flanked by piggyBac minimal inverted repeat elements.

14. A method for ing a composition comprising a homodimer and/or a multimer of the homodimer, wherein the homodimer comprises two monomers, each comprising amino acids 21-264 of SEQ ID NO:4, the method comprising: (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 ctor with ActiCHO P or ActiPro media at a growth temperature of 37 C ± 1; (c) g the cultures of (b) with HyClone Cell Boost 7a and HyClone Cell Boost 7b daily at a growth temperature of 37 C ± 1ºC until the cultures reach a cell density of about 10 million to about 15 million cells/mL; (d) shifting the growth temperature from 37 C ± 1ºC to 32.5ºC ± 1ºC; and (e) harvesting the mer and/or multimer of the homodimer from the culture media, n the method results in a final harvest protein titer of >3g/L of which >70% is present as the multimer of the homodimer, and wherein >28% of the multimers of the homodimer are heptamers of the mer or above.

15. The method of claim 14, wherein cell viability exceeds 85% at day 10, day 11, day 12, day 13, day 14, or longer of culture.

16. The method of claim 14 or claim 15, wherein greater than 80% of the composition comprises the multimer of the homodimer.

17. A method of purifying the homodimer and/or multimer of the homodimer produced by the method of any one of claims 6-11 comprising: (a) purifying the homodimer and/or er of the mer from the culture supernatant by affinity chromatography; and (b) polishing the homodimer and/or multimer of the homodimer by one or more of cation exchange chromatography, anion exchange chromatography, and hydrophobic interaction chromatography.

18. The method of claim 17, wherein the affinity chromatography uses a MabSelect SuRe protein A column.

19. The method of claim 17 or claim 18, wherein purification by affinity chromatography comprises eluting the homodimer and/or multimer of the homodimer from the affinity chromatography .

20. The method of claim 18, wherein the protein A column is regenerated with an NaOH buffer of at least 0.5M.

21. The method of any one of claims 17-20, wherein polishing the homodimer and/or multimer of the homodimer comprises anion ge flow through tography.

22. The method of claim 21, wherein anion exchange flow h chromatography comprises using a Q Sepharose Fast Flow column.

23. The method of any one of claims 17-22, wherein polishing the homodimer and/or er of the homodimer comprises cation exchange chromatography.

24. The method of claim 23, wherein cation exchange chromatography ses using a POROS XS .

25. The method of claim 23 or claim 24, wherein cation exchange chromatography comprises using a sodium acetate elution .

26. The method of claim 25, wherein the elution buffer further comprises 36.5-38.5% of a 1 M NaCl buffer.

27. The method of any one of claims 17-20, wherein polishing the homodimer and/or multimer of the homodimer comprises hydrophobic interaction chromatography.

28. The method of claim 27, n hydrophobic interaction chromatography comprises using a Butyl FF resin or Phenyl HP resin.

29. A method for purifying and polishing a ition comprising a mer and/or a multimer of the homodimer, wherein the homodimer comprises two monomers, each sing amino acids 21-264 of SEQ ID NO:4comprising: (a) purifying the homodimer and/or multimer of the homodimer from a culture supernatant by protein A affinity tography, wherein the protein A column uses an alkaline-resistant medium such as the MabSelect SuRe medium, wherein the ing is performed with at least two wash cycles; (b) polishing the homodimer and/or multimer of the homodimer by cation exchange chromatography, wherein the cation exchange column contains a high-capacity, highresolution resin such as POROS XS, and wherein the elution buffer is a sodium acetate buffer comprised of 36.5%-38.5% of a 1 M NaCl buffer; (c) polishing the homodimer and/or multimer of the homodimer by anion ge chromatography, n the anion exchange column contains an anion exchange medium such as a Q Sepharose Fast Flow medium; and (d) polishing the homodimer and/or multimer of the homodimer by hydrophobic interaction chromatography (HIC), wherein the HIC medium is a Butyl FF, a Phenyl HP or an Octyl FF resin and is selected to isolate or remove a particular fraction of the multimer of the homodimer in addition to polishing.

30 The method of claim 29, n the final protein titer of the composition is > 4 g/L.

31. The method of claim 29 or claim 30, wherein the composition comprises >70% multimers of the homodimer.

32. A composition comprising a recombinantly ed, purified homodimer and/or multimer of the homodimer made by the method of any one of claims 29-31.

33. Use of a inantly produced and purified homodimer and/or multimer of the homodimer made by the method of any one of claims 29-31 in the manufacture of a medicament for the treatment or prevention of an inflammatory, autoimmune, or infectious disease or disorder.

34. The method of claim 8, wherein the base culture media is ActiPro.

35. The method of claim 19, wherein the homodimer and/or multimer of the mer is eluted from the affinity chromatography column using an elution buffer comprising sodium acetate and NaCl.

36. The method of claim 14, wherein the final harvest protein titer is >3 g/L, >4 g/L, >5 g/L, >6 g/L, >7 g/L, >8 g/L, or >9g/L. mama“. . QTNE 22$ w .Su.