NZ795260A - Manufacturing Optimization of GL-2045, a Multimerizing Stradomer - Google Patents
Manufacturing Optimization of GL-2045, a Multimerizing StradomerInfo
- Publication number
- NZ795260A NZ795260A NZ795260A NZ79526017A NZ795260A NZ 795260 A NZ795260 A NZ 795260A NZ 795260 A NZ795260 A NZ 795260A NZ 79526017 A NZ79526017 A NZ 79526017A NZ 795260 A NZ795260 A NZ 795260A
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- homodimer
- composition
- multimer
- protein
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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).
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{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
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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.
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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).
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{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.
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{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
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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
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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;
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(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
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{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).
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{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.
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{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
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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
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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
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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).
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{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
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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
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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%
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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
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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.
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{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
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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
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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
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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
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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
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{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
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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
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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
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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,
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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%
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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%
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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
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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,
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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.
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} 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.
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{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
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(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
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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
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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
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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
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{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.
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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
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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—
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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,
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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
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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
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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
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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
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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
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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
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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
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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
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{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
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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
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(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
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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
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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
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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
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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).
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{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
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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
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{@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+ =
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(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
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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
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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
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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
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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
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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
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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).
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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 -
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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
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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
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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
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{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
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{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
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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
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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
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{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.
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{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
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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
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“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
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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
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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.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
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US62/432,402 | 2016-12-09 |
Publications (1)
Publication Number | Publication Date |
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NZ795260A true NZ795260A (en) | 2022-12-23 |
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