RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 62/095,361,
filed on December 22, 2014, which is incorporated by reference herein in its entirety.
TECHNICAL FIELD
The present disclosure relates to methods for selectively reducing CysL97 in a
preparation of IL-17 antibodies or antigen binding fragments thereof, e.g., a preparation of
secukinumab, that have been recombinantly produced by mammalian cells.
BACKGROUND OF THE DISCLOSURE
Classical antibodies are composed of two light chains (L) with a molecular weight of
about 25kD each and two heavy chains (H) with a molecular weight of about 50kD each. The
light and heavy chains are connected by a disulfide bond (L-S-S-H) and the two LH units are
further linked between the heavy chains by two disulfide bonds. The general formula of a
classical antibody is L-SS-H(-SS-)2H-SS-L or simply H2L2 (HHLL). Besides these conserved
inter-chain disulfide bonds, there are also conserved intra-chain disulfide. Both types of
disulfide bonds are important for the stability and behavior (e.g., affinity) of an antibody.
Generally, a disulfide bond is produced by two cysteine residues (Cys-SH) found at conserved
positions in the antibody chains, which spontaneously form the disulfide bond (Cys-S-S-Cys).
Disulfide bonds formation is determined by the redox potential of the environment and by the
presence of enzymes specialized in thiol-disulfide exchange. The internal disulfide bonds (Cys-
S-S-Cys) stabilize the three-dimensional structure of an antibody.
There are unusual antibodies that contain an additional free cysteine(s) (i.e., unpaired
cysteine) that is involved in antigen recognition and binding. For these antibodies, modification
of a free cysteine can have a negative effect on the activity and stability of the molecule, and can
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lead to increased immunogenicity. As a result, processing of these antibodies can be difficult, as
the end product may contain a substantial amount of inactive, misfolded and useless antibody
material. US20090280131, which is incorporated by reference herein in its entirety, provides IL-
17 antibodies, e.g., secukinumab (i.e., AIN457) with a free cysteine residue after the cis-proline
in the light chain complementarity determining region (CDR) 3 loop (L-CDR3) (i.e., amino acid
eight of L-CDR3 as set forth as SEQ ID NO:6, which corresponds to amino acid 97 of the light
chain variable region as set forth as SEQ ID NO:10, herein after referred to as “CysL97”). In
order to maintain full activity, the unpaired cysteine residue of secukinuamb cannot be masked
by oxidative disulfide pairing with other cysteine residues or by oxidation with exogenous
compounds (e.g., formation of mixed disulfides with other proteins, derivatization with cell
metabolites [e.g., cysteine or glutathione], and formation of sulfoxides by oxygen).
Unfortunately, because secukinuamb is manufactured using mammalian cells, undesired cellbased
modifications of CysL97 do occur.
The literature describes refolding of mammalian proteins expressed in bacterial cells,
which produce mammalian proteins as unfolded, insoluble aggregates having mixed disulfides
(inclusion bodies). To obtain mammalian proteins from bacteria, inclusion body proteins are
isolated, solubilized, and denatured with strong chaotropic reagents and reducing agents.
Complete denaturation and reduction of disulfide bonds using a denaturing agent, reducing
agent, disulfide adduct forming agent, and a mild oxidizing/reducing environment (pH 7-9) has
also been used to properly refold plant proteins obtained from commercial sources or
recombinantly produced in yeast (US 4,766,205). These processes, which employ complete
denaturation and refolding of proteins, are expensive, caustic, time-consuming, and unnecessary
for a protein produced in mammalian cells.
The use of reduction/oxidation coupling reagents to correct misfolding of non-naturally
occurring Fc fusion proteins is known (WO02/68455). The Fc fusion proteins of WO02/68455
presumably contain interchain disulfides in the Fc region that are reduced and reoxidzed by the
disclosed process, but there is no teaching therein of how to produce a molecule having a
selectively reduced cysteine residue. Moreover, an Fc fusion protein is simply not an antibody, a
highly complex immunoglobulin that relies on numerous properly linked inter-chain and intrachain
disulfides for structure and activity.
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US20050123532 provides methods of producing an antibody having a free cysteine by
activating the antibody with a reducing agent, or by culturing the antibody-producing cells in a
serum-free medium supplemented with L-cysteine. When using this cell-culture method, later
processing steps, e.g., filtration, viral inactivation and chromatography, could lead to oxidation
of the free cysteine produced by cell-culture methods. In such cases, the free cysteine is ideally
protected during later steps by modifying the free thiol group with an oxidizing agent, which is
itself later removed using various techniques, e.g., filtration (US20060257393). For commercial
production, such methods require large quantities of reducing agent in the original culture
medium, large quantities of an oxidizing agent during later processing, and additional filtration
methods to remove the oxidizing agent, adding time and expense to the cost of production.
U.S. Patent No. 7,928,205 teaches a preference for using redox pairs for refolding IgG2
antibodies obtained from mammalian cell cultures, as well as methods for decysteinylation of a
free cysteine in the variable region of the 146B7 (IgG1) antibody. The corresponding research
publication, Banks et al. (2008) J. Pharmaceutical Sci. 97:764-779, teaches decysteinylation of
an unpaired sulfhydryl in the variable region of a recombinant monoclonal IgG1 antibody
(MAB007). Banks et al. studied whether decysteinylation of MAB007 required the use of a
strong denaturant (GdnHCL) and a reducing agent (cysteine) or whether selective reduction
could occur in the presence of cysteine alone. The authors determined that cysteinylation was
effectively removed from MAB007 in the presence and absence of denaturant.
None of the above references teach whether selective reduction of CysL97 in secukinumab
is possible. Nor do the above references teach the reagents and conditions necessary for
selective reduction of CysL97 in secukinumab, which depend upon, inter alia, the primary,
secondary and tertiary structure of secukinumab; the position and location of oxidized CysL97 in
secukinumab (e.g., solvent-accessible or inaccessible); and the relative strength of the conserved
disulfide bonds in the antibody (e.g., whether CysL97 reacts first with a given reducing agent, or
only after conserved cysteines have been reduced). Moreover, none of these references describe
whether selective reduction of oxidized CysL97 in secukinumab would result in changes to the
antibody structure (e.g., folding), chemical composition (e.g., deamidation), or properties
(binding activity, propensity to aggregate or degrade), all of which could make it technically
unfeasible/impractical to selective reduce secukinumab at commercial scale.
5
SUMMARY OF THE DISCLOSURE
In order to maintain maximal secukinumab antigen-binding activity, we have determined
that it is necessary during processing of secukinumab to convert CysL97 from masked (1) to free
(2) form (see I, below) without significant reduction of the conserved disulfide bonds; otherwise
lower activity and inactive lower molecular weight variants will form by chain unlinking (H2L2
→ H2L, HL2, HL, H and L).
(I) H2L2-Cys-SX + reagent → H2L2-Cys-SH + X-reagent
(1) (2)
Introducing reducing conditions during commercial scale antibody preparation is
counterintuitive (see, e.g., Trexler-Schmidt et al. 2010 Biotech and Bioengineering 106:452-61,
which employs various reagents and methods to prevent antibody disulfide bond reduction
during cell culture manufacturing of antibodies). Nevertheless, we have determined that it is
possible to selectively reduce CysL97 in secukinumab during large scale commercial production
in mammalian cells without significant denaturation of the antibody. Disclosed herein are
methods for selectively reducing CysL97 in the antigen binding sites of the IL-17 antibodies (and
fragments thereof) disclosed in US20090280131, particularly secukinumab. These methods
assist in restoring the binding activity of these antibodies, and thus increase the bioactivity of
preparations thereof. Furthermore, these methods assist in increasing the level of intact antibody
and enhancing the homogeneity of preparations of these antibodies. The disclosed processes rely
on the combined effect of particular ratios of antibody:reductant and controlled oxygen transfer
rates in the system during incubation.
Accordingly, disclosed herein are methods for selectively reducing CysL97 in a
preparation of IL-17 antibodies that have been recombinantly produced by mammalian cells,
comprising:
a) contacting the preparation with at least one reducing agent in a system to form a
reducing mixture; and
b) incubating the reducing mixture while maintaining a volumetric oxygen mass-transfer
coefficient (kLa*) in the system of < about 0.37 h-1, said kLa* being calculated by
adapting a dissolved oxygen curve to a saturation curve;
wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable domain
(VH) comprising the three complementarity determining regions (CDRs) of the VH set forth as
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SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the three
CDRs of the VL set forth as SEQ ID NO:10, and further wherein prior to step a) the initial
percent oxygen saturation in the preparation is at least about 60%, as measured using an oxygen
probe calibrated at 25oC.
Also disclosed herein are methods for selectively reducing CysL97 in a preparation of IL-
17 antibodies that have been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with a set of oxidation/reduction reagents selected from
cysteine/cystine and cysteine/cystamine to form a reducing mixture; and
b) incubating the reducing mixture at a temperature of about 37 °C under anaerobic
conditions for at least about 4 hours, or incubating the reducing mixture at a temperature of about
18-24 °C for about 16-24 hours;
wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable
domain (VH) comprising the three complementarity determining regions (CDRs) of the VH set
forth as SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the
three CDRs of the VL set forth as SEQ ID NO:10.
Also disclosed herein are also purified preparations of secukinumab, wherein the level of
intact secukinumab in the preparation is at least about 90%, as measured by sodium dodecyl
sulfate capillary electrophoresis (CE-SDS), and wherein the level of activity of secukinumab in
the preparation is at least about 90%, as measured by cystamine-CEX.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1 shows the percentage of intact antibody (LHHL) over time after subjection to different
reducing agents.
Figure 2A shows the percentage of intact antibody (LHHL) over time at room temperature after
subjection to selective reduction with 8 mM cysteine under anaerobic conditions. Figure 2B
shows the percentage of intact antibody (LHHL) over time at room temperature after subjection
to selective reduction with 8 mM cysteine under aerobic conditions. Figure 2C shows the
percentage of intact antibody (LHHL) over time at 37°C after subjection to selective reduction
with 8 mM cysteine under anaerobic conditions. Figure 2D shows the percentage of intact
antibody (LHHL) over time at 37°C after subjection to selective reduction with 8 mM cysteine
under aerobic conditions.
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Figure 3 shows the percentage of intact antibody over time at 37 °C temperature after subjection
to selective reduction with 8 mM cysteine at various dissolved oxygen concentrations.
Figure 4A shows a 4D contour plot for activity by CEX. Figure 4B shows a 4D contour plot for
purity for CE-SDS. The plots of Figure 4 analyze the impact of and interaction of cysteine
concentration, protein content and ratio of cysteine/cystine on the output parameters activity by
CEX and purity by CE-SDS.
Figure 5A shows a 4D contour plot for activity by CEX. Figure 5B shows a 4D contour plot for
purity for CE-SDS. The plots of Figure 5 analyze the impact of and interaction of cysteine
concentration, protein content and cystine concentration on the output parameters activity by
CEX and purity by CE-SDS.
Figure 6 shows a 4D contour plot for activity by CEX, looking at the impact and interaction of
pH, time, temperature and cysteine concentration.
Figure 7 shows a 4D contour plot for purity by CE-SDS, looking at the impact and interaction of
pH, time, temperature and cysteine concentration.
Figure 8 shows the dissolved oxygen chart of the cysteine treatment of the confirmation run 1.
Figure 9 shows the dissolved oxygen chart of the cysteine treatment of the confirmation run 2.
Figure 10 shows the dissolved oxygen chart of the cysteine treatment of the confirmation run 3.
Figure 11 shows the dissolved oxygen chart of the cysteine treatment of the confirmation run 4.
Figure 12 compares the reaction kinetic of confirmation run 3 and confirmation run 4 with
respect to activity by CEX and purity by CE-SDS.
Figure 13A shows a scaled and centered coefficient plot for activity by CEX of REACT.P.
Figure 13B shows a 4D contour plot for activity by CEX of REACT.P. The plots of Figure 13
analyze the impact of and interaction of cysteine concentration, protein content and stirrer speed
on the output parameters activity by CEX and purity by CE-SDS.
Figure 14A shows the dissolved oxygen profiles of the process characterization runs at 0 rpm
(the numbers in the legend indicate run number, stirrer speed and cysteine and antibody
concentration). Figure 14B shows the dissolved oxygen profiles of the process characterization
runs at 50 rpm (the numbers in the legend indicate run number, stirrer speed and cysteine and
antibody concentration). Figure 14C shows the dissolved oxygen profiles of the process
characterization runs at 100 rpm (the numbers in the legend indicate run number, stirrer speed
and cysteine and antibody concentration). Figure 14D compares the dissolved oxygen profiles of
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the two runs performed at 50 mL scale at 50 rpm and 6.0 mM cysteine, one without antibody and
the other with 12.7 g/L antibody.
Figure 15 shows selective reduction kinetics of activity by CEX at different incubation
temperatures.
Figure 16 shows an overlay of dissolved oxygen charts from scale-down model qualification
runs, with an indication of the timing of various steps of the process (cysteine addition, heating,
incubation, cooling, and pH adjustment).
Figure 17 shows the kinetic for activity by CEX of the manufacturing-scale runs and the scaledown
model runs during selective reduction.
DETAILED DESCRPTION OF THE DISCLOSURE
It is an object of the disclosure to provide methods for selectively reducing CysL97 in the
antigen binding sites of certain IL-17 antibodies or antigen binding fragments thereof, such as
secukinumab. By “selectively reducing” is meant that CysL97 in a disclosed IL-17 antibody or
antigen binding fragment thereof is reduced to an oxidized form without reduction of the
conserved cysteine residues of these antibodies. The conserved cysteine residues, in the case of
a classical IgG1 antibody, are: two disulfide bridges in the hinge region, two inter-chain disulfide
bridges (one in each Fab), four intra-chain disulfide bridges in the Fc region, and eight intrachain
disulfide bridges in the Fab portion of the antibody. During the selective reduction
process, transient reduction of the conserved cysteines of some antibodies in a particular
preparation may occur. However, upon completion of the reaction, the vast majority of the
conserved cysteines that were transiently reduced will have reoxidized to form the conserved
disulfide bonds found in typical antibodies, resulting in high purity and activity in the selectively
reduced preparation (i.e., purified preparation) of antibodies. It will be understood that upon
completion of the selective reduction reaction, the selectively reduced preparation (i.e., purified
preparation) is not expected to contain 100% intact antibodies; instead the selectively reduced
preparation will ideally contain at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93
94, 95, 96, 97, 98, 99, or about 100 % (relative to theoretical maximum), intact antibodies as
measured by CE-SDS.
The term “comprising” encompasses “including” as well as “consisting” e.g. a
composition “comprising” X may consist exclusively of X or may include something additional
9
e.g. X + Y.
The term “about” in relation to a numerical value x means, for example, +/-10%. When
used in front of a numerical range or list of numbers, the term “about” applies to each number in
the series, e.g., the phrase “about 1-5” should be interpreted as “about 1 – about 5”, or, e.g., the
phrase “about 1, 2, 3, 4” should be interpreted as “about 1, about 2, about 3, about 4, etc.”
The relative molecular mass of secukinumab, based on post-translational amino acid
sequence, is 147,944 Daltons. This molecular weight (i.e., 147,944 Daltons) is used in the
calculation of secukinumab molarity values and molar ratios throughout the instant disclosure.
However, during production in CHO cells, a C-terminal lysine is commonly removed from each
heavy chain. The relative molecular mass of secukinumab lacking a C-terminal lysine from each
heavy chain is 147,688 Daltons. A preparation of secukinumab contains a mixture of molecules
with and without C-terminal lysine residues on the heavy chain. The secukinumab molarity
values (and ratios employing these molarity values) used in the instant disclosure are therefore
estimates, and the term “about”, “approximate” and the like in reference to these numerical
values encompasses at least this variation in relative molecular mass and the resulting
calculations made therewith.
The word “substantially” does not exclude “completely” e.g. a composition which is
“substantially free” from Y may be completely free from Y. Where necessary, the word
“substantially” may be omitted from the definition of the disclosure.
The term "antibody" as referred to herein includes whole antibodies and any antigenbinding
portion or single chains thereof. A naturally occurring "antibody" is a glycoprotein
comprising at least two heavy (H) chains and two light (L) chains inter-connected by disulfide
bonds. Each heavy chain is comprised of a heavy chain variable region (abbreviated herein as
VH) and a heavy chain constant region. The heavy chain constant region is comprised of three
domains, CH1, CH2 and CH3. Each light chain is comprised of a light chain variable region
(abbreviated herein as VL) and a light chain constant region. The light chain constant region is
comprised of one domain, CL. The VH and VL regions can be further subdivided into regions of
hypervariability, termed hypervariable regions or complementarity determining regions (CDR),
interspersed with regions that are more conserved, termed framework regions (FR). Each VH and
VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus
in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the
10
heavy and light chains contain a binding domain that interacts with an antigen. The constant
regions of the antibodies may mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g., effector cells) and the first
component (C1q) of the classical complement system.
The term "antigen-binding fragment" of an antibody as used herein, refers to fragments of
an antibody that retain the ability to specifically bind to an antigen (e.g., IL-17). It has been
shown that the antigen-binding function of an antibody can be performed by fragments of a fulllength
antibody. Examples of binding fragments encompassed within the term "antigen-binding
portion" of an antibody include a Fab fragment, a monovalent fragment consisting of the VL, VH,
CL and CH1 domains; a F(ab)2 fragment, a bivalent fragment comprising two Fab fragments
linked by a disulfide bridge at the hinge region; a Fd fragment consisting of the VH and CH1
domains; a Fv fragment consisting of the VL and VH domains of a single arm of an antibody; a
dAb fragment (Ward et al., 1989 Nature 341:544-546), which consists of a VH domain; and an
isolated CDR. Exemplary antigen binding sites include the CDRs of secukinumab as set forth in
SEQ ID NOs:1-6 and 11-13 (Table 1), preferably the heavy chain CDR3. Furthermore, although
the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be
joined, using recombinant methods, by a synthetic linker that enables them to be made as a single
protein chain in which the VL and VH regions pair to form monovalent molecules (known as
single chain Fv (scFv); see, e.g., Bird et al., 1988 Science 242:423-426; and Huston et al., 1988
Proc. Natl. Acad. Sci. 85:5879-5883). Such single chain antibodies are also intended to be
encompassed within the term "antibody”. Single chain antibodies and antigen-binding portions
are obtained using conventional techniques known to those of skill in the art.
An "isolated antibody", as used herein, refers to an antibody that is substantially free of
other antibodies having different antigenic specificities (e.g., an isolated antibody that
specifically binds IL-17 is substantially free of antibodies that specifically bind antigens other
than IL-17). The term "monoclonal antibody" or "monoclonal antibody composition" as used
herein refer to a preparation of antibody molecules of single molecular composition. The term
"human antibody", as used herein, is intended to include antibodies having variable regions in
which both the framework and CDR regions are derived from sequences of human origin. A
“human antibody” need not be produced by a human, human tissue or human cell. The human
antibodies of the disclosure may include amino acid residues not encoded by human sequences
11
(e.g., mutations introduced by random or site-specific mutagenesis in vitro, by N-nucleotide
addition at junctions in vivo during recombination of antibody genes, or by somatic mutation in
vivo). In some embodiments of the disclosed processes and compositions, the IL-17 antibody is
a human antibody, an isolated antibody, and/or a monoclonal antibody.
The term "IL-17" refers to IL-17A, formerly known as CTLA8, and includes wild-type IL-
17A from various species (e.g., human, mouse, and monkey), polymorphic variants of IL-17A,
and functional equivalents of IL-17A. Functional equivalents of IL-17A according to the present
disclosure preferably have at least about 65%, 75%, 85%, 95%, 96%, 97%, 98%, or even 99%
overall sequence identity with a wild-type IL-17A (e.g., human IL-17A), and substantially retain
the ability to induce IL-6 production by human dermal fibroblasts.
The term "KD" is intended to refer to the dissociation rate of a particular antibody-antigen
interaction. The term "KD", as used herein, is intended to refer to the dissociation constant, which
is obtained from the ratio of Kd to Ka (i.e. Kd/Ka) and is expressed as a molar concentration (M).
KD values for antibodies can be determined using methods well established in the art. A method
for determining the KD of an antibody is by using surface plasmon resonance, or using a
biosensor system such as a Biacore® system. In some embodiments, the IL-17 antibody or
antigen binding fragment, e.g., secukinumab, has a KD of about 100-250 pM for humanIL-17.
The term "affinity" refers to the strength of interaction between antibody and antigen at
single antigenic sites. Within each antigenic site, the variable region of the antibody “arm”
interacts through weak non-covalent forces with antigen at numerous sites; the more interactions,
the stronger the affinity. Standard assays to evaluate the binding affinity of the antibodies
toward IL-17 of various species are known in the art, including for example, ELISAs, western
blots and RIAs. The binding kinetics (e.g., binding affinity) of the antibodies also can be
assessed by standard assays known in the art, such as by Biacore analysis.
An antibody that "inhibits" one or more of these IL-17 functional properties (e.g.,
biochemical, immunochemical, cellular, physiological or other biological activities, or the like)
as determined according to methodologies known to the art and described herein, will be
understood to relate to a statistically significant decrease in the particular activity relative to that
seen in the absence of the antibody (or when a control antibody of irrelevant specificity is
present). An antibody that inhibits IL-17 activity affects a statistically significant decrease, e.g.,
by at least about 10% of the measured parameter, by at least 50%, 80% or 90%, and in certain
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embodiments of the disclosed methods and compositions, the IL-17 antibody used may inhibit
greater than 95%, 98% or 99% of IL-17 functional activity.
“Inhibit IL-6” as used herein refers to the ability of an IL-17 antibody or antigen binding
fragment thereof (e.g., secukinumab) to decrease IL-6 production from primary human dermal
fibroblasts. The production of IL-6 in primary human (dermal) fibroblasts is dependent on IL-17
(Hwang et al., (2004) Arthritis Res Ther; 6:R120-128). In short, human dermal fibroblasts are
stimulated with recombinant IL-17 in the presence of various concentrations of an IL-17 binding
molecule or human IL-17 receptor with Fc part. The chimeric anti-CD25 antibody Simulect
(basiliximab) may be conveniently used as a negative control. Supernatant is taken after 16 h
stimulation and assayed for IL-6 by ELISA. An IL-17 antibody or antigen binding fragment
thereof, e.g., secukinumab, typically has an IC50 for inhibition of IL-6 production (in the
presence 1 nM human IL-17) of about 50 nM or less (e.g., from about 0.01 to about 50 nM)
when tested as above, i.e., said inhibitory activity being measured on IL-6 production induced by
hu-IL-17 in human dermal fibroblasts. In some embodiments of the disclosed methods and
compositions, IL-17 antibodies or antigen binding fragments thereof, e.g., secukinumab, and
functional derivatives thereof have an IC50 for inhibition of IL-6 production as defined above of
about 20 nM or less, more preferably of about 10 nM or less, more preferably of about 5 nM or
less, more preferably of about 2 nM or less, more preferably of about 1 nM or less.
The term "derivative", unless otherwise indicated, is used to define amino acid sequence
variants, and covalent modifications (e.g., pegylation, deamidation, hydroxylation,
phosphorylation, methylation, etc.) of an IL-17 antibody or antigen binding fragment thereof,
e.g., secukinumab, according to the present disclosure, e.g., of a specified sequence (e.g., a
variable domain). A “functional derivative” includes a molecule having a qualitative biological
activity in common with the disclosed IL-17 antibodies. A functional derivative includes
fragments and peptide analogs of an IL-17 antibody as disclosed herein. Fragments comprise
regions within the sequence of a polypeptide according to the present disclosure, e.g., of a
specified sequence. Functional derivatives of the IL-17 antibodies disclosed herein (e.g.,
functional derivatives of secukinumab) preferably comprise VH and/or VL domains having at
least about 65%, 75%, 85%, 95%, 96%, 97%, 98%, or 99% overall sequence identity with the
VH and/or VL sequences of the IL-17 antibodies and antigen binding fragments thereof disclosed
13
herein (e.g., the VH and/or VL sequences of Table 1), and substantially retain the ability to bind
human IL-17 or, e.g., inhibit IL-6 production of IL-17 induced human dermal fibroblasts.
The phrase “substantially identical” means that the relevant amino acid or nucleotide
sequence (e.g., VH or VL domain) will be identical to or have insubstantial differences (e.g.,
through conserved amino acid substitutions) in comparison to a particular reference sequence.
Insubstantial differences include minor amino acid changes, such as 1 or 2 substitutions in a 5
amino acid sequence of a specified region (e.g., VH or VL domain). In the case of antibodies, the
second antibody has the same specificity and has at least 50% of the affinity of the same.
Sequences substantially identical (e.g., at least about 85% sequence identity) to the sequences
disclosed herein are also part of this application. In some embodiments, the sequence identity of
a derivative IL-17 antibody (e.g., a derivative of secukinumab, e.g., a secukinumab biosimilar
antibody) can be about 90% or greater, e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,
99% or higher relative to the disclosed sequences.
”Identity” with respect to a native polypeptide and its functional derivative is defined
herein as the percentage of amino acid residues in the candidate sequence that are identical with
the residues of a corresponding native polypeptide, after aligning the sequences and introducing
gaps, if necessary, to achieve the maximum percent identity, and not considering any
conservative substitutions as part of the sequence identity. Neither N- or C-terminal extensions
nor insertions shall be construed as reducing identity. Methods and computer programs for the
alignment are well known. The percent identity can be determined by standard alignment
algorithms, for example, the Basic Local Alignment Search Tool (BLAST) described by Altshul
et al. ((1990) J. Mol. Biol., 215: 403 410); the algorithm of Needleman et al. ((1970) J. Mol.
Biol., 48: 444 453); or the algorithm of Meyers et al. ((1988) Comput. Appl. Biosci., 4: 11 17).
A set of parameters may be the Blosum 62 scoring matrix with a gap penalty of 12, a gap extend
penalty of 4, and a frameshift gap penalty of 5. The percent identity between two amino acid or
nucleotide sequences can also be determined using the algorithm of E. Meyers and W. Miller
((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0),
using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
"Amino acid(s)" refer to all naturally occurring L--amino acids, e.g., and include Damino
acids. The phrase "amino acid sequence variant" refers to molecules with some
differences in their amino acid sequences as compared to the sequences according to the present
14
disclosure. Amino acid sequence variants of an antibody according to the present disclosure,
e.g., of a specified sequence, still have the ability to bind the human IL-17 or, e.g., inhibit IL-6
production of IL-17 induced human dermal fibroblasts. Amino acid sequence variants include
substitutional variants (those that have at least one amino acid residue removed and a different
amino acid inserted in its place at the same position in a polypeptide according to the present
disclosure), insertional variants (those with one or more amino acids inserted immediately
adjacent to an amino acid at a particular position in a polypeptide according to the present
disclosure) and deletional variants (those with one or more amino acids removed in a polypeptide
according to the present disclosure).
The phrases “free cysteine”, “non-traditional cysteine” and “unpaired cysteine”
interchangeably refer to a cysteine that is not involved in conserved antibody disulfide bonding.
The free cysteine may be present in an antibody framework region or a variable region (e.g.,
within a CDR). In secukinumab, amino acid eight of L-CDR3 as set forth as SEQ ID NO:6,
which corresponds to amino acid 97 of the light chain variable region as set forth as SEQ ID
NO:10 (herein after referred to as CysL97) is a free cysteine. Each molecule of secukinumab
comprises two such free cysteine residues – one in each VL domain. The disclosed processes are
capable of selectively reducing both free cysteine residues in secukinumab. In some
embodiments, e.g., due to deletions and/or substitutions in the light chain of a disclosed IL-17
antibody or antigen binding fragment thereof, the free cysteine will not be present at position
CysL97. In such case, the corresponding free cysteine is the target of the selective reduction
reaction and is included within the term “CysL97”.
IL-17 Antibodies and Antigen Binding Fragments Thereof
The various disclosed processes and relate to the selective reduction of certain IL-17
antibodies or antigen binding fragments thereof (e.g., secukinumab). In one embodiment, the IL-
17 antibody or antigen binding fragment thereof comprises at least one immunoglobulin heavy
chain variable domain (VH) comprising hypervariable regions CDR1, CDR2 and CDR3, said
CDR1 having the amino acid sequence SEQ ID NO:1, said CDR2 having the amino acid
sequence SEQ ID NO:2, and said CDR3 having the amino acid sequence SEQ ID NO:3. In one
embodiment, the IL-17 antibody or antigen binding fragment thereof comprises at least one
immunoglobulin light chain variable domain (VL’) comprising hypervariable regions CDR1’,
15
CDR2’ and CDR3’, said CDR1’ having the amino acid sequence SEQ ID NO:4, said CDR2’
having the amino acid sequence SEQ ID NO:5 and said CDR3’ having the amino acid sequence
SEQ ID NO:6. In one embodiment, the IL-17 antibody or antigen binding fragment thereof
comprises at least one immunoglobulin heavy chain variable domain (VH) comprising
hypervariable regions CDR1-x, CDR2-x and CDR3-x, said CDR1-x having the amino acid
sequence SEQ ID NO:11, said CDR2-x having the amino acid sequence SEQ ID NO:12, and
said CDR3-x having the amino acid sequence SEQ ID NO:13.
In one embodiment, the IL-17 antibody or antigen binding fragment thereof comprises at
least one immunoglobulin VH domain and at least one immunoglobulin VL domain, wherein: a)
the VH domain comprises (e.g., in sequence): i) hypervariable regions CDR1, CDR2 and CDR3,
said CDR1 having the amino acid sequence SEQ ID NO:1, said CDR2 having the amino acid
sequence SEQ ID NO:2, and said CDR3 having the amino acid sequence SEQ ID NO:3; or ii)
hypervariable regions CDR1-x, CDR2-x and CDR3-x, said CDR1-x having the amino acid
sequence SEQ ID NO:11, said CDR2-x having the amino acid sequence SEQ ID NO:12, and
said CDR3-x having the amino acid sequence SEQ ID NO:13; and b) the VL domain comprises
(e.g., in sequence) hypervariable regions CDR1’, CDR2’ and CDR3’, said CDR1’ having the
amino acid sequence SEQ ID NO:4, said CDR2’ having the amino acid sequence SEQ ID NO:5,
and said CDR3’ having the amino acid sequence SEQ ID NO:6.
In one embodiment, the IL-17 antibody or antigen binding fragment thereof comprises: a)
an immunoglobulin heavy chain variable domain (VH) comprising the amino acid sequence set
forth as SEQ ID NO:8; b) an immunoglobulin light chain variable domain (VL) comprising the
amino acid sequence set forth as SEQ ID NO:10; c) an immunoglobulin VH domain comprising
the amino acid sequence set forth as SEQ ID NO:8 and an immunoglobulin VL domain
comprising the amino acid sequence set forth as SEQ ID NO:10; d) an immunoglobulin VH
domain comprising the hypervariable regions set forth as SEQ ID NO:1, SEQ ID NO:2, and SEQ
ID NO:3; e) an immunoglobulin VL domain comprising the hypervariable regions set forth as
SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; f) an immunoglobulin VH domain comprising
the hypervariable regions set forth as SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; g) an
immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:1,
SEQ ID NO:2, and SEQ ID NO:3 and an immunoglobulin VL domain comprising the
hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6; or h) an
16
immunoglobulin VH domain comprising the hypervariable regions set forth as SEQ ID NO:11,
SEQ ID NO:12 and SEQ ID NO:13 and an immunoglobulin VL domain comprising the
hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6.
For ease of reference the amino acid sequences of the hypervariable regions of the
secukinumab monoclonal antibody, based on the Kabat definition and as determined by the Xray
analysis and using the approach of Chothia and coworkers, is provided in Table 1, below.
Light-Chain
CDR1’ Kabat R-A-S-Q-S-V-S-S-S-Y-L-A (SEQ ID NO:4)
Chothia R-A-S-Q-S-V-S-S-S-Y-L-A (SEQ ID NO:4)
CDR2’ Kabat G-A-S-S-R-A-T (SEQ ID NO:5)
Chothia G-A-S-S-R-A-T (SEQ ID NO:5)
CDR2’ Kabat Q-Q-Y-G-S-S-P-C-T (SEQ ID NO:6)
Chothia Q-Q-Y-G-S-S-P-C-T (SEQ ID NO:6)
Heavy-Chain
CDR1 Kabat N-Y-W-M-N (SEQ ID NO:1)
CDR1-x Chothia G-F-T-F-S-N-Y-W-M-N (SEQ ID NO:11)
CDR2 Kabat A-I-N-Q-D-G-S-E-K-Y-Y-V-G-S-V-K-G (SEQ ID NO:2)
CDR2-x Chothia A-I-N-Q-D-G-S-E-K-Y-Y (SEQ ID NO:12)
CDR3 Kabat D-Y-Y-D-I-L-T-D-Y-Y-I-H-Y-W-Y-F-D-L (SEQ ID NO:3)
CDR3-x Chothia C-V-R-D-Y-Y-D-I-L-T-D-Y-Y-I-H-Y-W-Y-F-D-L-W-G (SEQ ID
NO:13)
Table 1: Amino acid sequences of the hypervariable regions of the secukinumab monoclonal
antibodies.
17
In preferred embodiments, the constant region domains preferably also comprise suitable
human constant region domains, for instance as described in "Sequences of Proteins of
Immunological Interest", Kabat E.A. et al, US Department of Health and Human Services,
Public Health Service, National Institute of Health. DNA encoding the VL of secukinumab is set
forth in SEQ ID NO:9. DNA encoding the VH of secukinumab is set forth in SEQ ID NO:7.
In some embodiments, the IL-17 antibody or antigen binding fragment thereof (e.g.,
secukinumab) comprises the three CDRs of SEQ ID NO:10. In other embodiments, the IL-17
antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO:8. In
other embodiments, the IL-17 antibody or antigen binding fragment thereof comprises the three
CDRs of SEQ ID NO:10 and the three CDRs of SEQ ID NO:8. CDRs of SEQ ID NO:8 and
SEQ ID NO:10 may be found in Table 1. The free cysteine in the light chain (CysL97) may be
seen in SEQ ID NO:6.
In some embodiments, IL-17 antibody or antigen binding fragment thereof comprises the
light chain of SEQ ID NO:14. In other embodiments, the IL-17 antibody or antigen binding
fragment thereof comprises the heavy chain of SEQ ID NO:15 (with or without the C-terminal
lysine). In other embodiments, the IL-17 antibody or antigen binding fragment thereof
comprises the light chain of SEQ ID NO:14 and the heavy chain of SEQ ID NO:15 (with or
without the C-terminal lysine). In some embodiments, the IL-17 antibody or antigen binding
fragment thereof comprises the three CDRs of SEQ ID NO:14. In other embodiments, IL-17
antibody or antigen binding fragment thereof comprises the three CDRs of SEQ ID NO:15. In
other embodiments, the IL-17 antibody or antigen binding fragment thereof comprises the three
CDRs of SEQ ID NO:14 and the three CDRs of SEQ ID NO:15. CDRs of SEQ ID NO:14 and
SEQ ID NO:15 may be found in Table 1.
Hypervariable regions may be associated with any kind of framework regions, though
preferably are of human origin. Suitable framework regions are described in Kabat E.A. et al,
ibid. The preferred heavy chain framework is a human heavy chain framework, for instance that
of the secukinumab antibody. It consists in sequence, e.g. of FR1 (amino acid 1 to 30 of SEQ ID
NO:8), FR2 (amino acid 36 to 49 of SEQ ID NO:8), FR3 (amino acid 67 to 98 of SEQ ID NO:8)
and FR4 (amino acid 117 to 127 of SEQ ID NO:8) regions. Taking into consideration the
determined hypervariable regions of secukinumab by X-ray analysis, another preferred heavy
chain framework consists in sequence of FR1-x (amino acid 1 to 25 of SEQ ID NO:8), FR2-x
18
(amino acid 36 to 49 of SEQ ID NO:8), FR3-x (amino acid 61 to 95 of SEQ ID NO:8) and FR4
(amino acid 119 to 127 of SEQ ID NO:8) regions. In a similar manner, the light chain framework
consists, in sequence, of FR1’ (amino acid 1 to 23 of SEQ ID NO:10), FR2’ (amino acid 36 to 50
of SEQ ID NO:10), FR3’ (amino acid 58 to 89 of SEQ ID NO:10) and FR4’ (amino acid 99 to
109 of SEQ ID NO:10) regions.
In one embodiment, the IL-17 antibody or antigen binding fragment thereof (e.g.,
secukinumab) is selected from a human IL-17 antibody that comprises at least: a) an
immunoglobulin heavy chain or fragment thereof comprising a variable domain comprising, in
sequence, the hypervariable regions CDR1, CDR2 and CDR3 and the constant part or fragment
thereof of a human heavy chain; said CDR1 having the amino acid sequence SEQ ID NO:1, said
CDR2 having the amino acid sequence SEQ ID NO:2, and said CDR3 having the amino acid
sequence SEQ ID NO:3; and b) an immunoglobulin light chain or fragment thereof comprising a
variable domain comprising, in sequence, the hypervariable regions CDR1’, CDR2’, and CDR3’
and the constant part or fragment thereof of a human light chain, said CDR1’ having the amino
acid sequence SEQ ID NO: 4, said CDR2’ having the amino acid sequence SEQ ID NO:5, and
said CDR3’ having the amino acid sequence SEQ ID NO:6.
In one embodiment, the IL-17 antibody or antigen binding fragment thereof is selected
from a single chain antibody or antigen binding fragment thereof that comprises an antigen
binding site comprising: a) a first domain comprising, in sequence, the hypervariable regions
CDR1, CDR2 and CDR3, said CDR1 having the amino acid sequence SEQ ID NO:1, said CDR2
having the amino acid sequence SEQ ID NO:2, and said CDR3 having the amino acid sequence
SEQ ID NO:3; and b) a second domain comprising, in sequence, the hypervariable regions
CDR1', CDR2’ and CDR3’, said CDR1’ having the amino acid sequence SEQ ID NO:4, said
CDR2’ having the amino acid sequence SEQ ID NO:5, and said CDR3’ having the amino acid
sequence SEQ ID NO:6; and c) a peptide linker which is bound either to the N-terminal
extremity of the first domain and to the C-terminal extremity of the second domain or to the
C-terminal extremity of the first domain and to the N-terminal extremity of the second domain.
Alternatively, an IL-17 antibody or antigen binding fragment thereof as used in the
disclosed methods may comprise a derivative of the IL-17 antibodies set forth herein by
sequence (e.g., a pegylated version of secukinumab). Alternatively, the VH or VL domain of an
IL-17 antibody or antigen binding fragment thereof used in the disclosed methods may have VH
19
or VL domains that are substantially identical to the VH or VL domains set forth herein (e.g.,
those set forth in SEQ ID NO:8 and 10). A human IL-17 antibody disclosed herein may
comprise a heavy chain that is substantially identical to that set forth as SEQ ID NO:15 (with or
without the C-terminal lysine) and/or a light chain that is substantially identical to that set forth
as SEQ ID NO:14. A human IL-17 antibody disclosed herein may comprise a heavy chain that
comprises SEQ ID NO:15 (with or without the C-terminal lysine) and a light chain that
comprises SEQ ID NO:14. A human IL-17 antibody disclosed herein may comprise: a) one
heavy chain which comprises a variable domain having an amino acid sequence substantially
identical to that shown in SEQ ID NO:8 and the constant part of a human heavy chain; and b)
one light chain which comprises a variable domain having an amino acid sequence substantially
identical to that shown in SEQ ID NO:10 and the constant part of a human light chain.
Alternatively, an IL-17 antibody or antigen binding fragment thereof used in the
disclosed methods may be an amino acid sequence variant of the reference IL-17 antibodies set
forth herein, as long as it contains CysL97. The disclosure also includes IL-17 antibodies or
antigen binding fragments thereof (e.g., secukinumab) in which one or more of the amino acid
residues of the VH or VL domain of secukinumab (but not CysL97), typically only a few (e.g., 1-
10), are changed; for instance by mutation, e.g., site directed mutagenesis of the corresponding
DNA sequences. In all such cases of derivative and variants, the IL-17 antibody or antigen
binding fragment thereof is capable of inhibiting the activity of about 1 nM (= 30 ng/ml) human
IL-17 at a concentration of about 50 nM or less, about 20 nM or less, about 10 nM or less, about
5 nM or less, about 2 nM or less, or more preferably of about 1 nM or less of said molecule by
50%, said inhibitory activity being measured on IL-6 production induced by hu-IL-17 in human
dermal fibroblasts as described in Example 1 of WO 2006/013107.
In some embodiments, the IL-17 antibodies or antigen binding fragments thereof, e.g.,
secukinumab, bind to an epitope of mature human IL-17 comprising Leu74, Tyr85, His86,
Met87, Asn88, Val124, Thr125, Pro126, Ile127, Val128, His129. In some embodiments, the IL-
17 antibody, e.g., secukinumab, binds to an epitope of mature human IL-17 comprising Tyr43,
Tyr44, Arg46, Ala79, Asp80. In some embodiments, the IL-17 antibody, e.g., secukinumab,
binds to an epitope of an IL-17 homodimer having two mature human IL-17 chains, said epitope
comprising Leu74, Tyr85, His86, Met87, Asn88, Val124, Thr125, Pro126, Ile127, Val128,
His129 on one chain and Tyr43, Tyr44, Arg46, Ala79, Asp80 on the other chain. The residue
20
numbering scheme used to define these epitopes is based on residue one being the first amino
acid of the mature protein (ie., IL-17A lacking the 23 amino acid N-terminal signal peptide and
beginning with Glycine). The sequence for immature IL-17A is set forth in the Swiss-Prot entry
Q16552. In some embodiments, the IL-17 antibody has a KD of about 100-200 pM. In some
embodiments, the IL-17 antibody has an IC50 of about 0.4 nM for in vitro neutralization of the
biological activity of about 0.67 nM human IL-17A. In some embodiments, the absolute
bioavailability of subcutaneously (s.c.) administered IL-17 antibody has a range of about 60 –
about 80%, e.g., about 76%. In some embodiments, the IL-17 antibody, such as secukinumab,
has an elimination half-life of about 4 weeks (e.g., about 23 to about 35 days, about 23 to about
30 days, e.g., about 30 days). In some embodiments, the IL-17 antibody (such as secukinumab)
has a Tmax of about 7-8 days.
Particularly preferred IL-17 antibodies or antigen binding fragments thereof used in the
disclosed methods are human antibodies, especially secukinumab as described in Examples 1
and 2 of WO 2006/013107. Secukinumab is a recombinant high-affinity, fully human
monoclonal anti-human interleukin-17A (IL-17A, IL-17) antibody of the IgG1/kappa isotype
that is currently in clinical trials for the treatment of immune-mediated inflammatory conditions.
Secukinumab (see, e.g., WO2006/013107 and WO2007/117749) has a very high affinity for IL-
17, i.e., a KD of about 100-200 pM and an IC50 for in vitro neutralization of the biological
activity of about 0.67 nM human IL-17A of about 0.4 nM. Thus, secukinumab inhibits antigen
at a molar ratio of about 1:1. This high binding affinity makes the secukinumab antibody
particularly suitable for therapeutic applications. Furthermore, it has been determined that
secukinumab has a very long half-life, i.e., about 4 weeks, which allows for prolonged periods
between administration, an exceptional property when treating chronic life-long disorders, such
as rheumatoid arthritis.
Disclosed herein are processes for selectively reducing CysL97 in preparations of the
above-mentioned IL-17 antibodies and antigen binding fragments thereof (e.g., secukinumab).
The disclosed methods conveniently may be performed on preparations of antibodies (e.g., IL-17
antibodies, e.g., secukinumab) to reduce cost. A “preparation” of antibodies refers to a
composition (e.g., solution) having a plurality of an antibody molecule. A “preparation” includes
any liquid composition comprising the IL-17 antibody or antigen binding fragment thereof. As
such, a preparation may comprise, e.g., IL-17 antibody or antigen binding fragment thereof, e.g.,
21
secukinumab, in water or a buffer, in a column elutate, in a dialysis buffer, etc. In some
embodiments, the initial preparation of antibodies comprises a pool of the IL-17 antibodies or
antigen binding fragments thereof, e.g., secukinumab, in a buffer (e.g., a Tris, e.g., 1 mM – 1 M
Tris, pH 6.0-8.0) or WFI. Prior to addition of the reducing agent to the antibody, the preparation
may be adjusted by modifying dissolved oxygen levels, solution pH, antibody concentration, etc.
In some embodiments, prior to addition of a reducing agent, the concentration of antibody (e.g.,
secukinumab) in the preparation is adjusted to between about 4 mg/ml – about 19.4 mg/ml, e.g.,
about 10 mg/ml - about 19.4 mg/ml, about 10 mg/ml - about 15.4 mg/ml, about 12 mg/ml - about
15 mg/ml, or about 13.5 mg/ml. In some embodiments, prior to addition of the reducing agent,
the percent oxygen saturation in the preparation is adjusted to at least about 60% (as measured
using an oxygen probe calibrated at 25oC), e.g., at least about 80%. In some embodiments, prior
to addition of the reducing agent, the pH of the preparation is adjusted to about 7.3 - about 8.5,
e.g., about 7.8 - about 8.2, e.g., about 7.9 - about 8.1, e.g., about 8.0. The concentration of
antibody, pH and level of oxygen may also be adjusted immediately after (or even during)
addition of the reductant, and thus should be interpreted as equivalent.
The preparations of IL-17 antibodies or antigen binding fragments thereof for use in the
disclosed processes may be recombinantly produced by any mammalian cells using any
mammalian cell line, e.g., Chinese hamster ovary cells (CHO) cells, mouse myeloma NS0 cells,
baby hamster kidney (BHK) cells, human embryonic kidney cell line HEK-293, the human
retinal cell line Per.C6 (Crucell, NL), HKB11 cell clone (derived from a hybrid cell fusion of
HEK 293S with the Burkitt's lymphoma line 2B8), etc. By “recombinantly produced by
mammalian cells” is meant that production of the antibody in the mammalian cells has been
achieved using recombinant DNA technology. The IL-17 antibody preparation subjected to
selective reduction may be a pool of antibodies harvested from the mammalian cells by
centrifugation (with or without subsequent clarification). Alternatively, the IL-17 antibody
preparation subjected to selective reduction may be a pool of antibodies from a further
downstream chromatography step, e.g., an eluate from an affinity column (e.g., a protein A
column), a cation exchange column, an anion exchange column, etc. Alternatively, the IL-17
antibody preparation subjected to selective reduction may be a pool of antibodies from a
downstream filtration step, e.g., depth filtration, nanofiltration, ultrafiltaration, etc.
Alternatively, the IL-17 antibody preparation subjected to selective reduction may be a pool of
22
antibodies from a downstream step in which the pool has been treated to remove host cell
proteins and/or to inactivate viri. In a one embodiment, the preparation of IL-17 antibodies
subjected to selective reduction is a protein A eluate pool of antibodies.
Depending on the process conditions chosen, e.g., temperature, length of reaction time,
pH, etc.) the concentration of antibody in the original preparation will vary. In some
embodiments, the concentration of the IL-17 antibody used in the original preparation is between
about 2 mg/ml to about 20 mg/ml, about 3.8 mg/ml to about 19.5 mg/ml, about 4 mg/ml to about
19.5 mg/ml, about 10 mg/ml to about 19.4 mg/ml, e.g., about 10 mg/ml to about 15.4 mg/ml,
e.g., about 12 mg/ml to about 15 mg/ml, e.g., about 13.5 mg/ml of the IL-17 antibodies or
antigen binding fragments thereof. Prior to selective reduction, the antibody concentration in the
initial antibody preparation may be adjusted as desired using water for injection (WFI) or a
buffer of choice.
The selective reduction processes described herein may be performed in any size vessel.
In some embodiments, the vessel is lab-scale (e.g., 1L-2L). In other embodiments, the vessel is
pilot-scale (e.g., 12 L-20 L). In further embodiments, the vessel is commercial-scale (e.g.,
greater than 10,000 L, e.g., 14,000 L, 15,000 L, 16,000 L, etc.).
Reducing Agents
Reducing agents are substances capable of electron donation in a redox (reductionoxidation)
reaction. Specifically, such agents are useful to deliver hydrogen to a masked (or
blocked) cysteine present in the IL-17 antibody or antigen binding fragment thereof (e.g.,
secukinumab antibody). The process disclosed herein uses reducing agents for the selective
reduction of the IL-17 antibody. Each reducing agent referred to herein include derivatives
thereof (e.g., salts, esters and amides). Thus, e.g., reference to “cysteine” includes cysteine and
cysteine-HCL, reference to “TCEP” includes TCEP and TCEP-HCL, reference to thioglycolic
acid includes sodium thioglycolate, etc. Reducing agents for use in the disclosed methods
include sodium bisulfate, ammonia, triethylsilane, glycyclcysteine, sodium cyanoborohydride,
ammonium thioglycolate, calcium thioglycolate, sodium thioglycolate, ascorbic acid,
hydroquinone, aminomethanesulphonic acid, cysteic acid, cysteinesulphinic acid,
ethanedisulphonic acid, ethanesulphonic acid, homotaurine, hypotaurine, isethionic acid,
mercaptoethanesulphonic acid, N-methyltaurine (MTAU), TCEP (Tris(2-
23
carboxyethyl)phosphine hydrochloride), N-N-dimethyl-N-N bis(mercaptoacetyl)hydrazine
(DMH), dithiothreitol (DTT), 2-mercaptoethanol (beta-mercaptoethanol), 2-mercaptoacetic acid
(thioglycolic acid, TGA), cysteine (L-cysteine), cysteamine (beta-mercaptoethylamine, or
MEA), glutathione, and combinations thereof. In some embodiments, the reducing agent for use
in the disclosed process is a thiol-containing reducing agent (i.e., a compound having an R-SH
group), e.g., an organosulfur compound. In some embodiments, the reducing agent for use in the
disclosed process is, e.g., dithiothreitol (DTT), 2-mercaptoethanol, 2-mercaptoacetic acid,
cysteine, cysteamine, glutathione and combinations.
The strength of a reducing agent is indicated by its oxidation-reduction potential (redox
potential), Eo, which is given in Volts (V) and traditionally determined at pH 7, 25oC. For
example, the standard oxidation-reduction potential, Eo, for CSH/CSSC is given as about - 0.20
V to about -0.23 V (pH 7, 25oC) (P. C. Jocelyn (1967) Eu. J. Biochem 2:327-31; Liu “The role of
Sulfur in Proteins,” in The Proteins, 3rd Ed. (ed. Neurath) p. 250-252 Academic Press 1977).
The standard oxidation-reduction potential, Eo, of DTT is given as about -0.33 V (pH 7, 25oC)
(M.J.O'Neil, ed. by (2001). Merck Index: an encyclopedia of chemicals, drugs, & biologicals:
13th ed. (13. ed. ed.) United States: MERCK & CO INC.; Liu, supra). The standard oxidationreduction
potential, Eo, of glutathione is given as about about -0.24 V or about -0.26 V (pH 7,
25oC) (Rost and Rapoport (1964) Nature 201:185; Gilbert (1990) Adv. Enzymol. Relat. Areas
Mol. Biol. 63:69-172; Giles (2002) Gen. Physiol Biophys 21:65-72; Liu, supra). The standard
oxidation-reduction potential, Eo, of 2-mercaptoethanol is given as about -0.26 V (Lee and
Whitesides (1990) J. Org. Chem 58:642-647). In some embodiments, the reducing agent has a
standard oxidation-reduction potential, Eo, similar to cysteine (e.g., about -0.20 V to about -0.23
V, about -0.20 V to about -0.22 V, about -0.20 V to about -0.21 V, about -0.21 V to about -0.23
V, about -0.21 V to -0.22 V, about -0.22 V to about -0.23 V, about -0.20 V, about -0.21 V, about
-0.22 V, about -0.23 V).
The standard oxidation-reduction potential Eo of thiol-containing compounds may be
measured by thermal analysis, reduction of NAD+, polarography, reaction with Fe++, or thioldisulfide
exchange studies (Jocelyn, supra; Borsook et al. (1937) J. Biol. Chem 117:281; Ghosh
et al. (1932) J. Indian Chem. Soc. 9:43; Kolthoff et al. (1955) J. Am. Chem. Soc. 77:4739;
Tanaka et al. (1955) J. Am. Chem. Soc. 77:2004; Kolthoff et al. (1955) J. Am. Chem. Soc.
77:4733; Eldjarn (1957) J. Am. Chem. Soc. 79:4589). In some embodiments, the standard
24
oxidation-reduction potential Eo is determined by thermal analysis, polarography, reaction with
Fe++, or thiol-disulfide exchange studies, e.g., preferably by by thiol-disulfide exchange studies.
In some embodiments, the standard oxidation-reduction potential Eo is determined at pH 7, 25oC.
The reducing agent, when combined with the antibody preparation, forms a “reducing
mixture.” The reducing mixture may comprise excipients in addition to the reducing agent and
the IL-17 antibody. For example, in certain embodiments, a small molar ratio of the oxidized
form (e.g., cystine, cystamine) of the reducing agent may be added to the reducing mixture either
simultaneously with the reducing agent or sequentially, e.g., 10-30 minutes or more after the start
of incubation. For example, if cysteine is the reducing agent, than a small amount of cystine may
be added to the reducing mixture, e.g., concurrently with the cysteine or, e.g., 15, 20, 30 minutes
after cysteine is combined with the IL-17 antibody or antigen binding fragment thereof. Thus,
in some embodiments the reducing mixture comprises a set of oxidation/reduction reagents. By
“set of oxidation/reduction reagents” is meant a redox pair or redox couple, i.e., an oxidizing and
reducing agent that appear on opposite sides of a half-equation (e.g., a reducing species and its
corresponding oxidized form, e.g., Fe2+/Fe3+, cysteine/cystine, cysteamine/cystamine).
Depending on the reaction conditions (temperature, length of reaction time, quantity of
IL-17 antibody or antigen binding fragment thereof, pH, etc.) the concentration of reducing agent
used in a particular reducing mixture and selective reduction reaction will vary. In some
embodiments, the amount of reducing agent used in the reducing mixture will vary from about 1
to about 20 mM. In some embodiments, the concentration of reducing agent employed in the
reducing mixture is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15 or 16 mM. In one
embodiment, the amount of reducing agent (e.g., cysteine) is between about 4 mM and about 8
mM, e.g., 5.9 mM, 6 mM, 7.7 mM, 7.9 mM, 8 mM.
In one embodiment, the reducing agent is beta-mercaptoethanol. In certain embodiments,
beta-mercaptoethanol is employed at a concentration of about 2.0 mM to about 8.0 mM.
In one embodiment, the reducing agent is glutathione. In certain embodiments,
glutathione is employed at a concentration of about 2.0 mM to about 5.0 mM.
In one embodiment, the reducing agent is cysteamine. In certain embodiments,
cysteamine is employed at a concentration of about 1.0 mM to 20 mM, about 4.0 mM to about
19 mM, about 2.0 mM to about 8.0 mM, about 4.0 mM to about 8.0 mM, about 4.8 mM to about
8.0 mM, about 5.5 mM to about 6.7 mM, or about 6.0 mM.
25
In one embodiment, the reducing agent is cysteine. In certain embodiments, the
concentration of cysteine in the reducing mixture is about 1.0 mM to 20 mM, about 4.0 mM to
about 19 mM, about 2.0 mM to about 8.0 mM, about 4.0 mM to about 8.0 mM, about 4.8 mM to
about 8.0 mM, about 5.5 mM to about 6.7 mM, or about 6.0 mM. Cysteine concentration may
be adjusted using a stock solution of, e.g., 120 mM cysteine-HCL.
Each IL-17 antibody has two CysL97 residues in need of selective reduction. The
amount of reducing agent employed should therefore be sufficient to selectively reduce both
CysL97 residues on a substantial portion of IL-17 antibodies in a preparation of antibodies,
without concomitantly over-reducing the antibody by irreversibly reducing the traditional
disulfide bonds. Depending on the reaction conditions (presence of oxidizing agent, temperature,
length of reaction time, pH, etc.) the molar ratio of reducing agent:IL-17 antibody used in a
particular reducing mixture and selective reduction reaction will vary. We have found that the
molar ratio of reducing agent (e.g., cysteine):antibody (e.g., secukinumab) can range from about
11:1 (Example 5.2) to as high as about 546:1 (Example 6.2). In some embodiments of the
disclosed methods, the molar ratio of reducing agent (e.g., cysteine):antibody (e.g.,
secukinumab) is between about 11:1 to about 462:1 (e.g., about 21:1), about 31:1 to about 545:1
(e.g., about 31:1 to about 156:1), about 21:1 to about 296:1, or about 46:1 to about 91:1. In other
embodiments, the molar ratio of reducing agent (e.g., cysteine):antibody (e.g., secukinumab) is
between about 23:1 to about 91:1 (e.g., about 23:1 to about 57:1), about 44:1 to about 275:1
(e.g., about 44:1), about 44:1 to about 66:1 (e.g., about 44:1 to about 66:1), preferably about 46:1
to about 118:1 (e.g., about 56:1 to about 118:1), more preferably about 54:1 to about 82:1. In
one embodiment, the molar ratio of reducing agent (e.g., cysteine):antibody (e.g., secukinumab)
is about 66:1.
If a higher molar ratio of reducing agent (e.g., cysteine):antibody (e.g., secukinumab) is
used (representing excess reducing agent c.f. to antibody), then addition of a small amount of the
corresponding oxidizing agent (e.g., cystine or cystamine) may be useful to mitigate the
reductive power of the reducing agent (e.g., cysteine or cysteamine). This is particularly
beneficial in an anaerobic environment. Thus, in some embodiments, selective reduction is
carried out using a set of oxidation/reduction reagents (e.g., in an aerobic or anaerobic
environment, preferably an anaerobic environment). In some embodiments, selective reduction
is carried out using a molar ratio of reducing agent (e.g., cysteine):oxidizing agent (e.g., cystine)
26
of about 2:1 to about 80:1, about 4:1 to about 80:1, about 26:1 to about 80:1, about 2:1 to about
10:1 (e.g., about 6:1 to about 10:1), about 4:1 to about 28:1 (about 4:1 to about 18:1), about 27:1
to about 53:1 (e.g., about 27:1) in the reducing mixture. In certain embodiments, cysteine is used
in the reducing mixture in combination with the oxidizing agent cystine or cystamine (preferably
cystine). In some embodiments, selective reduction is carried out in conditions using about 4 mM
-14 mM cysteine (e.g., about 7.7 mM to about 8.0 mM cysteine) and about 0.1 to about 1 mM
cystine (e.g., about 0.1 to about 0.3 mM cystine) in the reducing mixture. In certain
embodiments, the reducing mixture contains about 8.0 mM cysteine and about 0.1 mM cystine,
about 7.9 mM cysteine and about 0.1 mM cystine, or about 7.7 mM cysteine and about 0.3 mM
cystine. It will be understood that if an oxidizing agent, e.g., cystine, is employed in
combination with the reducing agent, e.g., cysteine, in the disclosed process, the oxidizing agent,
e.g., cystine, may be added at a point after the reducing agent, e.g., cysteine, is combined with
the IL-17 antibody or antigen binding fragment thereof. For example, the IL-17 antibody or
antigen binding fragment thereof may be combined with cysteine to form a reducing mixture,
which is then incubated for, e.g., 15-30 minutes; thereafter, cystine may be added to the reaction.
Dissolved Oxygen
As used herein, “dissolved oxygen”, “dO2” and “DO” refer to the amount of oxygen that
is dissolved or carried in a given medium. It can be measured with an oxygen probe, such as an
oxygen sensor or an optode in liquid media. DO is reported as either as a concentration
(milligrams per liter (mg/L)) or as "percent saturation." Milligrams per liter is the amount of
oxygen in a liter of solvent and is also equivalent to parts per million = ppm. Percent oxygen
saturation is the amount of oxygen in a solution relative to the total amount of oxygen that the
solution can hold at a particular temperature.
As used herein, “initial percent oxygen saturation” refers to the amount of dissolved
oxygen in the preparation of IL-17 antibodies (e.g., secukinumab) prior to contacting the
preparation with the reducing agent in the vessel to form the reducing mixture. The initial
percent oxygen saturation can be adjusted directly (e.g., by sparging) or indirectly (e.g., by
stirring) to achieve a desired level of oxygen prior to the beginning of the selective reduction
process. For example, in some embodiments the initial percent oxygen saturation in the IL-17
antibody preparation is adjusted to at least 40%, 50%, 60%, 70%, 80%, 90%, or even as high as
27
100% prior to contact with the reducing agent. This may be done in order to initially mitigate the
power of the reducing agent once that agent is added to the IL-17 antibody preparation to form
the reducing mixture, which avoids partial or complete reduction of the traditional disulfides of
the antibody that otherwise would lead to loss of activity and purity. In preferred embodiments,
the initial percent oxygen saturation in the IL-17 antibody preparation is adjusted to at least 60%
(as measured using an oxygen probe calibrated at 25oC). In preferred embodiments, the initial
percent oxygen saturation in the IL-17 antibody preparation is adjusted to at least 80% (as
measured using an oxygen probe calibrated at 25oC).
We have determined that elevated oxygen levels during the cysteine treatment step can
have a deleterious effect on antibody activity, which is likely due to the oxygen abrogating the
reductive power of the cysteine, leading to insufficient reduction of C97 of secukinumab. This
issue of oxygen uptake from the atmosphere can be managed by varying the amount of reducing
agent, varying the molar ratio of reducing agent:antibody, using defined stirring speeds, or even
employing stirring interruptions, especially when working at production scale. It will be
understood that higher amounts of reducing agent (e.g., cysteine) are capable of handling higher
oxygen levels in the reducing mixture. In some embodiments, during the incubation step of the
selective reduction process, the oxygen saturation in the reducing mixture is generally
maintained at a low percentage (e.g., less than about 40%, less than about 30%, less than about
20%, less than about 15%, less than about 10%, less than about 5%).
A loss of reductive power of the reducing agent (e.g., cysteine) likely leads to incomplete
deblocking of CysL97-SH at earlier time points during the incubation step, and if there is no
residual reducing agent (e.g., cysteine) available to protect deblocked Cys97L at later portions of
the incubation step (or during the cooling step), then reoxidation of deblocked Cys97L-SH can
occur. Therefore, ideally, a low percentage oxygen saturation will be maintained for at least
about 60 minutes to about 330 minutes, e.g., at least about 60 minutes, at least about 90 minutes,
at least about 120 minutes, at least about 150 minutes, at least about 180 minutes, at least about
210 minutes, at least about 240 minutes, at least about 270 minutes, at least about 300 minutes,
or at least about 330 minutes. In some embodiments, this low percentage oxygen saturation will
be maintained for the full incubation step, as well as part of the cooling step.
Percent oxygen saturation can be adjusted directly (e.g., by sparging) or indirectly (e.g.,
by stirring) to achieve a desired level of oxygen during incubation of the reducing mixture. In an
28
aerobic environment, a low percent oxygen saturation may be achieved by using intermittent
(rather than continuous) mixing of the reducing solution, e.g., < 15 min/hr, e.g., <2 min/hr, or by
using continuous stirring with a low spin speed. In an anaerobic environment, no (or little)
oxygen is present that would lead to consumption of the reducing agent.
Volumetric oxygen mass-transfer (kLa*)
When selective reduction is performed under aerobic conditions, the level of oxygen in
the reaction is not controlled directly, but via other process parameters, e.g., stir speed. The
physical setup of each reaction also influences the level of oxygen present in the reaction
mixture. Therefore, it is important to identify a variable that can be used to compare the oxygen
transferred into a solution between physical setups and during particular antibody processing
steps – that variable is “kLa*” (see, e.g., Garcia-Ochoa and Gomez (2009) Biotechnology
Advances 27:153-176; Bandino et al. (2001) Biochem. Engineering J. 8:111-119; Juarez and
Orejas (2001) Latin Am. Appl. Res. 31:433-439; Yange and Wang (1992) Biotechnol. Prog.
8:244-61). The kLa* represents the amount of oxygen transferred into a solution over time via the
headspace without sparging. This value is specific for each setup and scale, and depends on
stirrer type, stirrer speed, filling volume and surface area of the solution in contact with the
headspace, which is influenced by the individual geometry of each vessel. While the kLa* of
each physical setup differs, because the level of oxygen in the solution during the selective
reduction process effects the activity and integrity of secukinumab, we expect that the selective
reduction process, when performed in systems displaying similar kLa* ranges, will lead to
preparations of secukinumab having similar quality.
As used herein the term “system” encompasses both the physical setup (vessel, stir type,
etc.) and the process conditions (fill volume, spin speed, etc.) that influence the oxygen transfer
into a solution over time via the headspace without sparging, i.e., scale, stirrer speed, filling
volume, surface area of the solution in contact with the headspace, which is influenced by the
individual geometry of each vessel, etc.
As used herein the term “vessel” means any container in which the selective reduction
reaction takes place. Vessels include, without limitation, bioreactors (e.g., steel, stirred tank,
disposable or non-disposable, etc.) used for pilot and commercial scale antibody production, as
well as common laboratory containers, such as flasks, tubes, etc. In some embodiments, the
29
vessel is a bioreactor capable of holding a volume of at least about 2 liters, at least about 100
liters, at least about 500 liters, at least about 1000 liters, at least about 2000 liters, at least about
5000 liters, at least about 10,000 liters, at least about 15,000 liters or greater.
The kLa* cannot be directly determined in the oxygen transfer experiments. Instead, the
dO2 in a test solution is replaced by nitrogen and the increase of dO2 over time is monitored
using a calibrated dO2 probe, which allows creation of an experimental dO2 curve. Thereafter, the
kLa* values used herein are calculated for the particular systems by adapting the experimental
dO2 curve to a saturation curve (e.g., using Mathcad®) according the equation shown below:
(1 ) kLa* (t t0) DO C e       , where DO = the measured value of dissolved oxygen, C is the
saturation value of oxygen (meaning 100 % when stirred infinitely and saturation is
achieved), Euler’s number e = 2.718281…., t = time point corresponding to the DO
value, and t0 = starting time point.
The equation represents the integrated form of an empirical formula established for
determination of the oxygen transfer into solutions (kLa* value). The formula was confirmed by
different authors in various experiments (Doran, P. M. 1995. Bioprocess Engineering Principles,
Academic Press, San Diego, California).
We have determined that the heating and cooling steps of the selective reduction process
are generally more tolerable of a higher kLa* than the incubation step. This is because elevated
oxygen levels during the incubation step can have a deleterious effect on antibody activity,
which is likely due to the increased oxygen transfer abrogating the reductive power of the
reducing agent (e.g., cysteine), leading to insufficient reduction of C97 of secukinumab. In an
aerobic environment, increasing the stir speed (or stir time) in the reducing mixture during the
selective reduction process increases the kLa*. Continuous stirring of the reducing mixture,
which leads to higher kLa* values, can be tolerated during the heating and cooling steps. In some
embodiments, the kLa* during the heating and cooling steps (separately) can be from about 0.12
h-1 to about 1.69 h-1, about 0.08 h-1to about 0.69 h-1, about 0.24 h-1 to about 0.44 h-1, about 0.39 h-
1 to about 0.69 h-1. In some embodiments, the kLa* in the system during the heating or cooling
step is < about 0.69 h-1, said kLa* being calculated by adapting a saturation curve to a dissolved
oxygen curve.
It is preferable to keep the kLa* lower during the incubation step using, e.g., continuous
stirring with low spin speeds, or, preferably, intermittent stirring. In some embodiments, the
30
reducing mixture is incubated while maintaining a volumetric oxygen mass-transfer coefficient
(kLa*) in the system of < about 0.37 h-1, < about 0.27 h-1, or < about 0.18 h-1, said kLa* being
calculated by adapting a dissolved oxygen curve to a saturation curve. If the kLa* in the system
during the incubation step of the selective reduction reaction is less than (<) 0.37 h-1, then the
molar ratio of reducing agent (e.g., cysteine):antibody can vary between 46.11:1 (about 46:1) to
118.36:1 (about 118:1) (for both shorter and longer incubation times, e.g., about 210 to about
330 minutes). The kLa* in the system during the incubation step of the selective reduction
reaction can be as high as (<) 0.37 h-1 if the molar ratio of reducing agent (e.g., cysteine):protein
is between 69.89:1 (about 70:1) to 118.36:1 (about 118:1) (for shorter incubation times, e.g., up
to about 240 minute incubation) or between 76.85 (about 77:1) to 118.36:1 (about 118:1) (for
longer incubation times, e.g., up to about 300 minute incubation).
The entire selective reduction process (i.e., heating step, incubation step, and cooling
step) can be generally performed using a kLa* of < 0.37 h-1, which includes about 210 - about
330 minute incubation time (e.g., about 240 minute - 300 minute incubation time), if the molar
ratio of reducing agent (e.g., cysteine):antibody is between about 46:1 - about 118:1. The entire
selective reduction process (i.e., heating step, incubation step, and cooling step) can be generally
performed using a kLa* < 0.37 h-1 if the molar ratio of reducing agent (e.g., cysteine):antibody is
between about 70:1 - about 118:1, and incubation is up to about 240 minutes; or if the molar
ratio of reducing agent (e.g., cysteine):antibody is between about 77:1 - about 118:1, and
incubation is up to about 300 minutes.
Further Process Components
The present disclosure provides a method for selective reduction of CysL97 in certain IL-
17 antibodies, such as secukinumab, comprising contacting the antibody with a reducing agent to
form a reducing mixture. It will be understood that the reducing mixture may comprise
components in addition to the IL-17 antibody or antigen binding fragment thereof and the
reducing agent. The reducing mixture may contain an aqueous component (e.g., a buffer, such as
a Tris buffer), as well as reagents used to increase or decrease the pH of the reducing mixture,
salt, EDTA, etc. Thus, while the reducing mixture will necessarily contain the IL-17 antibody
(e.g., secukinumab) and the reducing agent, the reducing mixture may or may not contain
additional components. In some embodiments, the reducing mixture contains a metal chelator
31
(e.g, EDTA, DMSA, DMPS). In some embodiments, the reducing mixture contains 1.3 mM to
about 0.8 mM, about 1.1 to about 0.9 mM, or about 1.0 mM EDTA (e.g., di-Na-EDTA).
Depending on the reaction conditions (temperature, length of reaction time, quantity of
IL-17 antibody, concentration of reducing agent, ratio reducing agent:antibody, etc.) the pH of a
particular antibody preparation may vary. However, it is recognized that, based upon the
reaction conditions described herein, such conditions can be varied in order to achieve the
desired selective reduction. In some embodiments, the pH of the antibody preparation prior to
contact with the reducing agent will vary from about 6.5 to about 9.5, e.g., about 7 to about 9. In
other embodiments, the pH of the antibody preparation will be about 7.4 to about 8.5, about 7.8
to about 8.2, about 7.9 to about 8.1, or about 8.0. In some embodiments, the pH of the antibody
preparation may be adjusted (e.g., following gassing to adjust the initial percent oxygen
saturation) using a buffer, e.g., a 1M Tris buffer (e.g., 1M Tris buffer pH 10.8).
Following contacting of the preparation containing the IL-17 antibody or antigen binding
fragment thereof (e.g., secukinumab) with the reducing agent, there may be an initial reduction in
the level of intact IL-17 antibody or antigen binding fragment thereof (HLLH). The term
“intact” refers to an antibody having all conserved disulfide bridges (e.g., 14 conserved disulfide
bridges in the case of a classical IgG1 antibody). The formation of various IL-17 antibody
fragments, i.e., H2L, HL2, HL, H and L bands, as well as the intact H2L2 (HHLL) band can be
determined using different analytical tools known in the art (such as SDS-PAGE, Cationexchange
HPLC). Preferably, the level of intact IL-17 antibody or antigen binding fragment
thereof in the mixture is measured by sodium dodecyl sulfate capillary electrophoresis (CESDS).
CE-SDS separates proteins according to their molecular size in an electric field. Nonreducing
CE-SDS can be used to assess size variants in a preparation of antibodies. In some
embodiments, the level of intact IL-17 antibody or antigen binding fragment thereof in the
mixture decreases to at least about 80%, as measured by CE-SDS, within about 1-30 minutes
(e.g., about 15 minutes) of addition of the reducing agent to the antibody preparation. In some
embodiments, the level of intact IL-17 antibody or antigen binding fragment thereof in the
mixture decreases to at least about 83%, as measured by CE-SDS. In some embodiments, the
level of intact IL-17 antibody or antigen binding fragment thereof in the mixture decreases to
between about 75% to about 87%. In some embodiments, the level of intact IL-17 antibody or
antigen binding fragment thereof in the mixture decreases to at least about 38, 39, 40, 41, 45, 50,
32
55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, or 87%, as measured
by CE-SDS.
CE-SDS analyses may be performed using a Beckman Coulter PA-800 capillary
electrophoresis system. Uncoated fused-silica capillaries with an inner diameter of 50 μm and a
length of 30 cm (with 20 cm and 10 cm separation ranges for reducing CE-SDS and nonreducing
CE-SDS analyses, respectively) are used for the analyses. The separation is monitored
with a UV detector at 214 nm. For non-reducing CE-SDS analyses, antibody samples are diluted
to 6.0 mg/mL with water, mixed thoroughly with non-reducing CE-SDS sample buffer (0.1 M
Tris/1.0% SDS, pH 7.0) and 250 mM iodoacetamide, at a ratio of 20/75/5 (v/v/v), and heated at
70°C for 10 minutes to prevent disulfide bridge shuffling. The capillary temperature is set at
25°C for the separation. The electrophoresis is carried out at a constant voltage of 15 kV in the
normal polarity mode for 20 minutes.
In some embodiments, the reducing mixture is heated. In some embodiments, heating
occurs prior to the step of incubating. In some embodiments, the reducing mixture is heated to a
temperature between about 32oC to about 42oC, to between about 35oC to about 39oC, to about
37oC. In some embodiments, the heating occurs for about 30 to about 120 minutes, about 45 to
about 90 minutes, about 45 to about 75 minutes, about 60 minutes. During heating, the reducing
mixture may be stirred, e.g., constantly or intermittently, using any means for stirring. Stirring
may be axial (e.g., using a pitched blade impeller) or radial (e.g., using a rushton turbine). In
some embodiments, during heating the reducing mixture is constantly stirred at 65-200 rpm (e.g.,
50 rpm, 65 rpm, 75 rpm, 85 rpm, 100 rpm or 200 rpm).
During the incubating step, the reducing mixture will typically be incubated for a
predetermined time to allow selective reduction of the free cysteine (e.g., the free cysteine of
secukinumab). In some embodiments, incubation occurs following heating of the reducing
mixture. Depending on the reaction conditions (reducing agent, temperature, quantity of IL-17
antibody or antigen binding fragment thereof, pH, etc.) the predetermined time for incubation of
the reducing mixture will vary. In some embodiments, the time will vary between about 1 and
24 hours (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 16, 18, 20 or 24 hours). In some
embodiments, incubating is performed for about 200 to about 500 minutes, about 210 to about
420 min., about 210 to about 330 minutes, about 240 to about 300 minutes, about 250 minutes.
During the incubating step, incubation will be performed at a predetermined temperature
33
to allow selective reduction of the target free cysteine (e.g., CysL97 in the antigen binding sites
of secukinumab). Depending on the reaction conditions (reducing agent, time, quantity of IL-17
antibody or antigen binding fragment thereof, pH, etc.) the incubation temperature will vary. In
some embodiments, the predetermined temperature will vary between about 20 to about 42oC. In
some embodiments, the predetermined temperature will be about 32oC - about 42oC, between
about 35oC - about 39oC, or about 37oC.
During the incubating step, the reducing mixture may be stirred to ensure product
homogeneity while the reductant is incubated with the IL-17 antibody preparation, e.g.,
secukinumab. However, the level of oxygen in the vessel should be kept low during this portion
of the selective reduction reaction in order to allow the reducing agent to effectively selectively
reduce CysL97 in the IL-17 antibodies. Under aerobic conditions, low oxygen may be achieved
by avoiding continuous stirring, e.g., by using intermittent stirring, e.g., < 15 min/hr, e.g., < 2
min/ hr. Under anaerobic conditions, transfer of oxygen is limited and therefore stirring may
proceed for longer periods of time or may be continuous. Moreover, under anaerobic conditions,
strict control of oxygen transfer (e.g., by regulated sparging) would also allow application of
longer periods of stir time (including continuous stirring).
In some embodiments, the mixture is cooled following the incubating step. In some
embodiments, the mixture is cooled to room temperature (e.g., a temperature between about
16oC to about 28oC). In some embodiments, cooling occurs for about 30 to about 120 minutes,
about 45 to about 90 minutes, about 45 to about 75 minutes, about 60 minutes. During cooling,
the reducing mixture may be stirred, e.g., constantly or intermittently using any means for
stirring. Stirring may be axial (e.g., using a pitched blade impeller) or radial (e.g., using a
rushton turbine). In some embodiments, during cooling the reducing mixture is constantly
stirred at 65-200 rpm (e.g., 50 rpm, 65 rpm, 75 rpm, 85 rpm, 100 rpm or 200 rpm).
The selective reduction reaction may be quenched, e.g., using iodoacetamid or ophosphoric
acid (e.g., using a stock solution of 0.3 M o-phosphoric acid). In some
embodiments, quenching occurs following the cooling step. In some embodiments, the selective
reduction reaction is quenched by adjusting the pH of the mixture to between about 5.0 to about
5.5, about 5.1 to about 5.3, about 5.2. pH adjustment may be achieved using o-phosphoric acid.
The phrase “purified preparation” refers to a mixture of IL-17 antibodies or antigen
binding fragments thereof that have been subjected to selective reduction. After completion of
34
selective reduction, there will be an increase in the level of intact IL-17 antibody in the purified
preparation. The level of intact antibodies in the purified preparation after selective reduction
may be measured via various well known techniques (e.g., non-reducing SDS PAGE, CE-SDS
PAGE, size exclusion chromatography (SEC), HPLC). In some embodiments, the level of intact
antibody is measured by CE-SDS. In some embodiments, after completion of selective reduction
(e.g., after the cooling step), the level of intact IL-17 antibody or antigen binding fragment
thereof in the purified preparation is at least about 80%, as measured by CE-SDS. In some
embodiments the level of intact IL-17 antibody or antigen binding fragment thereof in the
purified preparation is at least about 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93 94, 95,
96, 97, 98, 99, or about 100 %, as measured by CE-SDS, after selective reduction. In some
embodiments, the level of intact IL-17 antibody or antigen binding fragment thereof in the
purified preparation is at least about 90%, as measured by CE-SDS, after selective reduction.
The activity (e.g., affinity, biological activity, etc.) of the antibodies in a preparation prior
to selective reduction, during selective reduction or after selective reduction (i.e., in a purified
preparation) may be measured via various well known techniques (see, e.g., WO2006/013107;
WO2007/117749; Shen and Gaffen (2008) Cytokine. 41(2): 92-104). In certain embodiments,
the activity is measured using an ELISA based assay or a cell-based assay (e.g., inhibition of IL-
17 dependent release of IL-6 or GROalpha from, e.g., C-20/A4 chondrocytes or BJ cell line). In
some embodiments, activity is measured by a cystamine-CEX (cation exchange
chromatography) method. The cystamine-CEX method includes derivatization of the antibody
with cystamine (2,2’-dithiobis(ethylamine)), followed by analytical separation using cation
exchange chromatography (CEX). Because the activity of the antibodies disclosed herein (e.g.,
secukinumab) is decreased if CysL97 is in oxidized form, derivatization of CysL97 with
cystamine serves as a proxy to measure antibody activity, i.e., if selective reduction succeeds
then reduced CysL97 can be derivitized with cystamine, whereas if selective reduction fails then
oxidized CysL97 cannot be derivitized with cystamine. Derivatization by cystamine leads to an
addition of one positive charge per free Cys97 residue. The resulting derivatized forms of
secukinumab (e.g., +2, +1 charges) can then be separated from the non-derivatized form and
quantified by CEX. A cystamine-derivatized secukinumab molecule with two cystamine bound
to unpaired Cys97 on both light chains may be considered 100% biological active in theory. A
cystamine-derivatized secukinumab molecule with addition of one cystamine bound to unpaired
35
Cys97 on one of the light chains may be considered 50% biological active. A cystaminederivatized
secukinumab molecule without any cystamine bound to the molecule may be
considered biological inactive. The level of cystamine derivitization in a preparation of
antibodies (e.g., a preparation of secukinumab antibodies), in comparison to the theoretical
maximum level of cystamine derivitization in that preparation (e.g., expressed as a percentage of
theoretical maximum) may then be used as a measure of the activity of the preparation.
In brief cystamine-CEX may be performed as follows. Antibody samples (50 μg) are
first treated with carboxypeptidase B (1:40, w:w) to remove the C-terminal lysine in the heavy
chain and then derivatized with 4 mM cystamine in 5 mM sodium acetate, 0.5 mM EDTA, pH4.7
at room temperature for 2 hours. The derivatization is stopped by addition of 2 μL of 1M
phosphoric acid. CEX is performed on the cystamine-derivatized antibody samples using a
ProPac™ WCX-10 analytical column (4 mm x 250 mm, Dionex). A gradient from 12.5 mM to
92.5 mM sodium chloride in 25 mM sodium phosphate, pH 6.0 at a flow rate of 1.0 ml/min is
used for separation. Absorption at 220 nm is recorded by a UV detector (Agilent HPLC 1200).
Some initial preparations of IL-17 antibody with an oxidized free cysteine have activity
levels as low as 45%. In some embodiments, prior to initiating the selective reduction process,
the level of activity of the IL-17 antibodies or antigen binding fragments thereof in the
preparation is less than about 80%, less than about 75%, less than about 70%, less than about
65%, less than about 60%, less than about 55%, less than about 50%, or less than about 45%
(e.g., as measured by the cystamine-CEX method). During selective reduction, there will be an
increase in the level of activity of the IL-17 antibodies in the purified preparation. In some
embodiments, the level of activity of the IL-17 antibodies or antigen binding fragments thereof
in the antibody preparation increases by at least about 15 percentage points (e.g., from about
60% to at least about 75%), at least about 20 percentage points (e.g., from about 60% to at least
about 80%), at least about 25 percentage points (e.g., from about 60% to at least about 85%) or at
least about 30 percentage points (e.g., from about 60% to at least about 90%) within about 60
minutes following contacting the antibody preparation with the reducing agent to form the
reducing mixture (e.g., as measured by the cystamine-CEX method).
After selective reduction, the purified preparation will be enriched for IL-17 antibodies
having the reduced form of CysL97 and will display an increased level of activity relative to the
initial preparation. In some embodiments, after completion of selective reduction, the level of
36
activity of the IL-17 antibodies or antigen binding fragments thereof in the purified preparation is
at least about 80% (relative to a theoretical maximum), as measured by cystamine-CEX, an
ELISA, or cell-based binding assay (e.g., a cystamine-CEX assay). In some embodiments, after
completion of selective reduction, the level of activity of the IL-17 antibodies or antigen binding
fragments thereof in the purified preparation is at least about 80, 81 82, 83, 84, 85, 86, 87, 88, 89,
90, 91, 92, 93 94, 95, 96, 97, 98, 99 or about 100 %, as measured by CEX, an ELISA, or cellbased
binding assay (e.g., a cystamine-CEX assay). In some embodiments, after completion of
selective reduction, the level of activity of the IL-17 antibodies or antigen binding fragments
thereof in the purified preparation is at least about 93%, as measured by cystamine-CEX, an
ELISA, or cell-based binding assay (e.g., a cystamine-CEX assay).
Accordingly, disclosed herein are methods for selectively reducing CysL97 in a
preparation of IL-17 antibodies that have been recombinantly produced by mammalian cells,
comprising:
a) contacting the preparation with at least one reducing agent in a system to form a
reducing mixture; and
b) incubating the reducing mixture while maintaining a volumetric oxygen mass-transfer
coefficient (kLa*) in the system of < about 0.37 h-1, said kLa* being calculated by
adapting a dissolved oxygen curve to a saturation curve;
wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable domain
(VH) comprising the three complementarity determining regions (CDRs) of the VH set forth as
SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the three
CDRs of the VL set forth as SEQ ID NO:10, and further wherein prior to step a) the initial
percent oxygen saturation in the preparation is at least about 60%, as measured using an oxygen
probe calibrated at 25oC.
Also disclosed herein are methods for selectively reducing CysL97 in a preparation of IL-
17 antibodies that have been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with a set of oxidation/reduction reagents selected from
cysteine/cystine and cysteine/cystamine to form a reducing mixture; and
b) incubating the reducing mixture at a temperature of about 37 °C under anaerobic
conditions for at least about 4 hours, or incubating the reducing mixture at a temperature of about
18-24 °C for about 16-24 hours;
37
wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable
domain (VH) comprising the three complementarity determining regions (CDRs) of the VH set
forth as SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the
three CDRs of the VL set forth as SEQ ID NO:10.
Also disclosed herein are methods for selectively reducing CysL97 in a preparation of
secukinumab antibodies that have been recombinantly produced by mammalian cells,
comprising:
a) adjusting the concentration of secukinumab in the preparation to between about 4
mg/ml – about 19.4 mg/ml, e.g., about 10 mg/ml - about 19.4 mg/ml, e.g., about 10 - about 15.4,
e.g., about 12 mg/ml - about 15 mg/ml, e.g., about 13.5 mg/ml;
b) adjusting the percent oxygen saturation in the preparation to at least about 60%,
e.g., at least about 80%;
c) adjusting the pH of the preparation to about 7.4 - about 8.5, e.g., about 7.8 -
about 8.2, e.g., about 7.9 - about 8.1, e.g., about 8.0;
d) contacting the preparation with cysteine in a vessel to form a reducing mixture,
wherein the concentration of cysteine in the reducing mixture is about 4.0 mM - about 8.0 mM,
e.g., about 4.8 mM - about 8.0 mM, e.g., about 5.5 mM - about 6.7 mM, e.g., about 6.0 mM;
e) heating the reducing mixture to a temperature between about 32oC - about 42oC,
e.g., to between about 35oC - about 39oC, e.g., to about 37oC, said heating occurring for about 45
- about 90 minutes, e.g., about 45 - about 75 minutes, e.g., about 60 minutes;
f) incubating the reducing mixture from step e) at a temperature between about 20oC
- about 42oC, e.g., 32oC - about 42oC, e.g., to between about 35oC - about 39oC, e.g., to about
37oC, said incubating occurring for about 210 - about 420 minutes, e.g., about 210 - about 330
minutes, e.g., about 240 - about 300 minutes, e.g., about 250 minutes while maintaining a
volumetric oxygen mass-transfer coefficient (kLa*) in the vessel of < 0.37 h-1, said kLa* being
calculated by adapting a saturation curve to a dissolved oxygen curve,
g) cooling the mixture resultant from step f) to a temperature between about 16oC -
about 28oC, said cooling occurring for about 45 - about 90 minutes, e.g., about 45 - about 75
minutes, e.g., about 60 minutes; and
h) adjusting the pH of the mixture resultant from step g) to between about 5.1 - about
5.3, e.g., about 5.2.
38
Also disclosed herein are also purified preparations of secukinumab, wherein the level of
intact secukinumab in the preparation is at least about 90%, as measured by sodium dodecyl
sulfate capillary electrophoresis (CE-SDS), and wherein the level of activity of secukinumab in
the preparation is at least about 90%, as measured by cystamine-CEX.
General
In some embodiments of the above methods, the IL-17 antibody or antigen binding
fragment thereof comprises: i) an immunoglobulin heavy chain variable domain (VH)
comprising the amino acid sequence set forth as SEQ ID NO:8; ii) an immunoglobulin light
chain variable domain (VL) comprising the amino acid sequence set forth as SEQ ID NO:10; iii)
an immunoglobulin VH domain comprising the amino acid sequence set forth as SEQ ID NO:8
and an immunoglobulin VL domain comprising the amino acid sequence set forth as SEQ ID
NO:10; iv) an immunoglobulin VH domain comprising, in sequence, the hypervariable regions
set forth as SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3; v) an immunoglobulin VL domain
comprising, in sequence, the hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5 and
SEQ ID NO:6; vi) an immunoglobulin VH domain comprising, in sequence, the hypervariable
regions set forth as SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13; vii) an
immunoglobulin VH domain comprising, in sequence, the hypervariable regions set forth as SEQ
ID NO:1, SEQ ID NO:2, and SEQ ID NO:3 and an immunoglobulin VL domain comprising, in
sequence, the hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6;
and viii) an immunoglobulin VH domain comprising, in sequence, the hypervariable regions set
forth as SEQ ID NO:11, SEQ ID NO:12 and SEQ ID NO:13 and an immunoglobulin VL domain
comprising, in sequence, the hypervariable regions set forth as SEQ ID NO:4, SEQ ID NO:5 and
SEQ ID NO:6. In some embodiments of the disclosed methods, the IL-17 antibody or antigen
binding fragment thereof is a human antibody of the IgG1 isotype. In some embodiments of the
disclosed methods, the antibody is secukinumab.
The details of one or more embodiments of the disclosure are set forth in the
accompanying description above. Although any methods and materials similar or equivalent to
those described herein can be used in the practice or testing of the present disclosure, the
preferred methods and materials are now described. Other features, objects, and advantages of
the disclosure will be apparent from the description and from the claims. In the specification and
39
the appended claims, the singular forms include plural referents unless the context clearly
dictates otherwise. Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ordinary skill in the art to which this
disclosure belongs. All patents and publications cited in this specification are incorporated by
reference. The following Examples are presented in order to more fully illustrate the preferred
embodiments of the disclosure. These examples should in no way be construed as limiting the
scope of the disclosed patient matter, as defined by the appended claims.
EXAMPLES
Example 1: Reduction of Secukinumab Protein A intermediate by different sulfhydryl
agents
Example 1.1
A variety of sulfhydryl group-containing reducing agents (e.g., dithiothreitol (DTT), 2-
mercaptoethanol, 2-mercaptoacetic acid, cysteine, cysteamine, glutathione) were screened for
their use in selectively reducing the oxidized Cys97 in the light chain of secukinumab. In a first
set of experiments, 1-mL portions of inactive starting material obtained from an early
fermentation process after the Protein A capture step were incubated at 37°C at various pH with
various concentrations of β-mercaptoethanol, cysteine and glutathione. After certain time-points,
the samples were desalted by gel filtration into 20mM acetate pH 6 buffer and restoration of
activity was determined by an ELISA. Also, the content of free sulfhydryl groups was
determined by Ellman test (deblocking of the sulfhydryl group should result in a value of 2 Mol
free-SH per mol antibody). The results are listed in the Table 2, below.
2-ME° Activity Free SH Cysteine Activity Free SH Glutathione Activity Free SH
% * Mol/Mol % * Mol/Mol % * Mol/Mol
pH 7
37°C 5mM/1h 59 1.3
/4h 85 1.6
/8h 85 1.6
pH8
37°C 1mM/2h 61 1.4
/8h 90 1.8
2mM/1h 64 1.4 2mM/2h 80 1.8 2mM/2h 45 1.4
/4h 82 1.9 /4h 98 1.9 /8h 65 2.0
/8h 88 n.p.
40
4mM/1h 88 1.3 4mM/1h 77 0.9 4mM/2h 59 1.6
/4h 91 1.8 /4h 105 1.9 /4h 94 1.8
/8h 106 2.0
8mM/1h 58 1.4
/4h 75 1.7
pH 9
37°C 1mM/2h 48 1.2
/8h 71 1.5
2mM/1h 82 n.p. 2mM/2h 78 1.6 2mM/2h 73 1.6
/4h 97 0.9 /4h 89 1.8 /8h 81 2.1
/8h 93 1.1
4mM/1h 85 0.7 4mM/1h 74 1.5 4mM/2h 54 2.0
/4h 102 2.0 /4h 100 2.1 /4h 73 2.1
8mM/1h 68 1.7
/4h 81 2.1
Table 2: Activity and free-SH of samples following reaction at given conditions.
°: 2-Mercaptaoetanol (2-ME)
*: % relative to reference
As a result of these studies, mercaptoethanol and cysteine in the pH range 7-9 at
concentration of 1 to 20 mM and temperatures of 20-40°C were found to be most suitable.
In the course of further investigations, it was realized that antibody can be overreduced
during exposure to the reducing agents with formation of antibody having partially reduced
interchain disulfide bridges. This overreduction is reversible at atmospheric conditions when the
antibody is isolated from the reaction mixture, e.g., by diafiltration as described above or by
chromatography, because dissolved oxygen present in the buffers leads to spontaneous reoxidation
with re-formation of intact antibody.
L-SS-HH-SS-L → L-SH + HS-HH-SS-L → L-SS-HH-SS-L
(intact antibody) (reduced interchain disulfide bridge) (intact antibody)
Example 1.2 (anaerobic conditions)
In a second set of experiments, the treatments of secukinumab antibody obtained after the
Protein A capture step were performed under anaerobic conditions by using de-aerated solutions
and argon or nitrogen atmosphere to exclude effects of dissolved oxygen on the reduction
outcome. In brief, a secukinumab solution from a Protein A capture step was adjusted to pH 8.0
by addition of a Tris base stock solution. The solution was then adjusted to a concentration of 8
41
mM cysteine and 1 mM EDTA by addition of a cysteine/EDTA stock solution (e.g.,
200mM/12.5 mM EDTA at pH 8). The concentration of secukinumab in the reducing solution
was 4 mg/mL (molar ratio of cysteine:antibody about 296:1). The mixture was incubated at
room temperature for a period of 24 hours. At different time-points, samples were drawn and
spiked with an excess of iodoacetamid, which stops the reaction by quenching sulhydryl groups
of the reducing agent and antibody.
The same set-up was used for the experiments with 2-mercaptoethanol, 2-mercaptoacetic
acid, cysteine, cysteamine and glutathione. For DTT, a concentration of 1mM was used.
The quenched samples were analyzed by capillary electrophoreses with SDS in the nonreduced
mode (CE-SDS). This analytical method separates the different reduction products of
antibody (HHL, HH, HL, H and L species) from intact antibody (LHHL) and quantifies them by
on-line UV detection and area-integration of the obtained signals.
In Figure 1, the data for intact antibody (LHHL) from 0, 3, 15 min, 1h, 2h, 4h and 20-24h
reduction treatment shows that DTT and mercapto acetic acid reduce the antibody completely
without re-equilibration to intact antibody. Glutathione and mercaptoethanol also show
pronounced reduction, but were able to induce a re-equilibration to intact antibody (LHHL).
Cysteine reduces the antibody to about 50%, which is followed by a straight-forward reequilibration
to intact antibody. The data for cysteamine shows that this reagent either exerts
little reduction or leads very quickly (within a time of less than 3min) to re-equilibration.
The reduction order found (i.e., DTT > β-mercapto acetic acid > β-mercaptoethanol >
glutathione) correlates to published data for redox potentials or disulfide interchange. see T. Liu
in “The Proteins, 3rd Edition, Volume 3 (1977) p. 239 which gives following comments and
data:
Redox potential E = E0 + 0.059 x log [R-SS-R]/[R-SH]
Standard redox potential (E0 in Volt) not directly measurable as electrode is poised by the sulfur.
Therefore, redox potential had to be deduced from indirect measurements:
Dithiothreitol DTT/DTTox : E0 ~ -0.33 V (pH 7, 25°C)
Glutathione GSH/GSSG : E0 ~ -0.24 V (pH 7, 25°C)
Cysteine CSH/CSSC : E0 ~ -0.22 V (pH 7, 25°C)
42
Example 2: Cysteine Selectively Reduces Secukinumab Under Aerobic and Anaerobic
Conditions.
Additional experiments were performed at room temperature (RT) (about 25oC) or 37°C
using 8 mM cysteine, 4 mg/ml secukinumab, pH 8 (molar ratio of cysteine:antibody about
295.88:1). Reactions were performed under either aerobic (i.e., preparation of the solutions and
the treatment was carried out under normal air) or anaerobic (i.e., preparation of the solutions
and the treatment was carried out under exclusion or reduction of oxygen by de-aeration and
subsequent working under argon or nitrogen atmosphere) conditions.
Under anaerobic conditions (e.g., nitrogen or argon atmosphere with 0% dissolved oxygen),
in the early phase of the treatment there is a substantial degradation of antibody with formation
of antibody fragments - a sign of over-reduction. The maximal decrease of intact antibody
(LHHL) to about 40% occurs at around 15 minutes for experiments performed at either RT
(Figure 2A) or 37°C (Figure 2C). Re-equilibration to intact antibody at 20 hours shows about
17.1% over-reduced variants remaining in the RT samples and 12.1% over-reduced variants
remaining in the 37°C samples.
Under aerobic conditions, i.e., in presence of dissolved oxygen, there is a more moderate
reaction. The maximal decrease of intact antibody (LHHL) to about 80% residual level occurs at
around 15 minutes for experiments performed at RT (Figure 2B) and to about 73% for
experiments performed at 37oC (Figure 2D). Re-equilibration to intact antibody shows only
5.8% over-reduced variants remaining in the RT samples after 20 hours and only 9.3% overreduced
variants remaining in the 37oC samples after 8 hours.
Thus, under anaerobic conditions the initial reduction is larger and re-equilibration to intact
antibody is not as complete as under aerobic conditions.
Example 3: Influence of Dissolved Oxygen and Cystine on Selective Reduction
Because cysteine (Cys-SH) oxidizes in presence of air to cystine (Cys-SS-Cys), the
difference observed under aerobic and anaerobic conditions could be caused by small amounts of
cystine formed during preparation of cysteine solutions under aerobic conditions and also during
the actual treatment of secukinumab with cysteine. In order to assess the influence of the level of
43
dO2 and cystine (or cystamine) on selective reduction of secukinumab, we performed several
additional preparative experiments with isolation of the antibody.
For some samples, selective reduction of 4 mg/ml secukinumab was performed with 8
mM cysteine (molar ratio of cysteine:antibody 295.88:1) in a solution having a pH 8.0 at 37oC
under aerobic conditions at 100% dO2 (no fumigation by nitrogen), 50%, and 20% dO2 (stirring
at ambient atmosphere with fumigation by nitrogen such that dissolved oxygen stayed at target
level throughout the experiment), and anaerobic (0% dO2) conditions (degassed solutions, full
nitrogen atmosphere) without cystine, as well as under 100% dO2 in presence of about 0.1 mM
cystine [ratio cysteine:cystine = 80:1]. In one experiment, 0.3 mM cystine was added following
an initial 30 minute incubation with 8 mM cysteine under anaerobic conditions. In another
experiment, selective reduction was performed under anaerobic conditions using 7.7 mM
cysteine and 0.3 mM cystine [ratio cysteine:cystine = 25.66:1]. In another sample, 0.1 mM
cystamine was added instead of cystine.
In all these examples, samples were drawn at different time points. At the end of the
treatment (240 min at 37°C), the antibody was isolated by adjusting the pH of the bulk solution
to 5.0 and loading onto a cation-exchange column that binds the antibody. After a wash to
remove the reducing agent, antibody was eluted by a salt and/or pH-gradient and analyzed by
CE-SDS, SE-HPLC and CEX.
Figure 3 shows CE-SDS analysis of iodacetamid quenched samples drawn at different
time of the treatment. Less dO2 leads to greater reduction in the early phase and slower
equilibration in the later phase of selective reduction. Level of intact antibody was substantially
lower (70%) at the end of treatment in total anaerobic conditions (0% dO2). Level of intact
antibody was improved to above 90% when the reaction was performed under conditions of 50%
or more dissolved oxygen. Addition of a small amount of cystine (or cysteamine) decreased the
initial reduction of antibody.
Table 3 shows activity and purity of antibody obtained after the samples from different
reactivation treatments (anaerobic and aerobic conditions) were purified using subsequent
chromatography on SP-Sepharose FF.
Treatment conditions after SP- step
(37°C/pH 8.0/4h) CE-SDS
purity
SEC
purity
CEX
activity
Bioactivity
44
% % % %
Starting material 96.4 98.4 61.2 45
8mM cysteine/ 0% oxygene (anaerob) 82.3 98.1 92.8 92
8mM cysteine/0% oxygene /30min: + 0.3mM cystine 92.6 98.4 92.3 95
8mM cysteine/ 20% oxygene 96.9 98.0 88.6 99
8mM cysteine/ 50% oxygene 96.1 98.2 87.0 90
8mM cysteine/100% oxygene (aerob) 95.8 98.1 90.6 96
7.7mM cysteine/0.3mM cystine/anaerob 97.1 98.9 91.8 104
8mM cysteine/0.1mM cystine 100% oxygene (aerob) 94.5 98.0 82.4 108
8mM cysteine/0.1mM cystamine 100% oxygene (aerob) 94.8 98.6 86.1 86
Table 3: Activity and purity of antibody following different reactivation treatments.
With respect to bioactivity, in all cases fully active material was obtained (86-108 % of
theoretical maximum) versus the non-selectively reduced secukinumab starting material, which
had only 45% activity. With respect to CE-SDS purity, impaired purity (82.3%) was found in
antibody obtained under completely anaerobic treatment lacking cystine, which was probably
due to over-reduction under these conditions (note: addition of cystine to anaerobic reactions
increased the purity of the samples as measured by CE-SDS, because reduction potential is
smaller and by this over-reduction less likely). However, all other selective reduction treatments
led to similar, and high, antibody quality and activity. We also noted that some CE-SDS purity
values prior to the SP-Sepharose chromatography step were slightly lower than the values
obtained following the chromatography step (e.g., 65% before vs 82% after) (data not shown),
which suggests that additional re-oxidation may occur during the chromatography step. CEX
activity was generally lower under the various aerobic conditions because there was more
oxygen to mitigate the reductive power of the cysteine. Addition of the oxidative agents cystine
and cystamine under aerobic conditions further decreased CEX activity.
Summary and Conclusions Drawn from Examples 1-3
We have determined that CysL97 of secukinumab is available for reduction in solution,
without requiring partial unfolding of the full antibody structure, e.g., using guanidine HCl.
Moreover, secukinumab overall is amenable to selective reduction, which, under controlled
conditions, should allow activation of the antibody without substantial degradation.
Cysteine was found to be particularly ideal for selective reduction of secukinumab, as it
45
displayed only moderate over-reduction, coupled with fast equilibrium. In the first hour of
selective reduction using cysteine, the antibody is partially reduced, e.g., up to 60% under
anaerobic conditions and up to about 30% under fully-aerobic conditions. Subsequently,
secukinumab slowly re-oxidizes to intact antibody. Re-oxidation of samples subjected to
selective reduction with cysteine proceeded much more slowly at room temperature than
reactions performed at 37oC (c.f. 21 hours for re-equilibrium at room temperature versus 8 hrs
for equilibrium at 37oC). Room temperature samples also generally displayed smaller maximal
decreases in intact antibody compared to selective reduction reactions performed at 37oC.
Under anaerobic conditions, we noted that the initial reduction of the antibody is larger
and re-equilibration to intact secukinumab is not as complete as under aerobic conditions.
However, addition of a small amount of cystine to anaerobic reactions resulted in improved
purity and activity relative to anaerobic reactions performed in the absence of cystine. The
aerobic reaction course can thus be simulated under anaerobic conditions when a small molar
ratio of the oxidized from of the reducing reagent (e.g., cystine in the case of cysteine as the
reducing agent) is present. Moreover, we noted that addition of cystine accelerated equilibrium
of intact antibody – even when cystine was not present during the initial 30 minutes of
incubation. Thus, selective reduction may be performed in the presence of air, as well as in the
absence of dissolved oxygen by using an inert gas atmosphere (e.g., nitrogen or argon). If
performed under fully anaerobic conditions, addition of a small molar ratio of the oxidized form
of the reducing reagent is useful.
Example 4: Cysteine Selective Reduction Step Development Study: Proof of Concept
(DoE1)
Example 4.1 - Study Design and Methods
The main purpose of the cysteine treatment step is to regain the full biological activity of
the secukinumab antibody by the masking of the –SH group of the cysteine in position 97 of the
light chain, which can occur during cell cultivation, harvesting and / or Protein A
chromatography. In the following Example, the purity of the antibody is analyzed by a nonreducing
CE-SDS method that monitors antibody integrity (called “purity by CE-SDS”) and the
biologic activity of the antibody is analyzed by a cystamine-CEX chromatography method
(called “activity by CEX”). In cystamine-CEX chromatography, cystamine is added to the
46
antibody sample and allowed to derivatize any free cysteine in secukinumab. The sample is then
subjected to analytical separation by cation exchange chromatography, whereby cystaminederivatized
antibody species elute after non-derivatized antibody species because they carry an
additional positive charge. The chromatogram is then compared to the chromatogram of the
non-treated sample. The portion of species in the chromatogram of the treated sample having a
shifted elution position is a direct measure of the abundance of free CysL97-SH in the original
sample and can be used as surrogate marker for activity.
The main purpose of the proof of concept study (DoE1) was to check the applicability of
design of experiments studies for improving of the cysteine treatment step to test a first set of
parameter ranges to identify main influencing factors and support definition of operating ranges.
Additionally, DoE1 was performed to evaluate if the experimental setup is applicable to detect
the influence of input parameters on output parameters. The concept study was analyzed using
Modde 9.0 (Umetrics) Software.
The input parameters are shown in Table 4. The addition of cystine (oxidized form) was
tested as an additional input parameter, as the redox potential is influenced by the ratio reduced
form/oxidized form, in this case the ratio cysteine/cystine. The ratio cysteine/cystine and cysteine
concentration were investigated on 3 levels each and the antibody concentration (“content by
ALC”) on 2 levels. For product quality output parameters, activity by CEC and purity by CESDS
were determined.

WE CLAIM:
1. A method for selectively reducing CysL97 in a preparation of IL-17 antibodies that have
been recombinantly produced by mammalian cells, comprising:
c) contacting the preparation with at least one reducing agent in a system to form a
reducing mixture; and
d) incubating the reducing mixture while maintaining a volumetric oxygen mass-transfer
coefficient (kLa*) in the system of < about 0.37 h-1, said kLa* being calculated by
adapting a dissolved oxygen curve to a saturation curve;
wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable domain
(VH) comprising the three complementarity determining regions (CDRs) of the VH set forth as
SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the three
CDRs of the VL set forth as SEQ ID NO:10; and wherein prior to step a) the initial percent
oxygen saturation in the preparation is at least about 60%, as measured using an oxygen probe
calibrated at 25oC.
2. The method according to claim 1, wherein the at least one reducing agent is selected from
the group consisting of cysteamine, cysteine, and combinations thereof.
3. The method according to claim 1, wherein the at least one reducing agent is a thiolcontaining
reducing agent having a standard oxidation-reduction potential, Eo, of about -0.20 V
to about -0.23 V at pH 7.0, as measured by thiol-disulfide exchange.
4. The method according to claim 2, wherein the at least one reducing agent is cysteine.
5. The method according to claim 4, wherein the kLa* in the system during step b) is <
about 0.37 h-1, wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is
between about 56:1 to about 118:1, and wherein the reducing mixture is incubated according to
step b) for up to about 240 minutes.
6. The method according to claim 4, wherein the kLa* in the system during step b) is <
103
about 0.37 h-1, wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is
between about 77:1 to about 118:1, and wherein the reducing mixture is incubated according to
step b) for up to about 300 minutes.
7. The method according to claim 4, wherein the kLa* in the system during step b) is < about
0.37 h-1, and wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is
between about 46:1 to about 118:1.
8. The method according to claim 4, wherein the kLa* in the system during step b) is < about
0.37 h-1, and wherein the molar ratio of cysteine:IL-17 antibodies in the reducing mixture is
between about 54:1 to about 82:1.
9. The method according to claim 4, wherein the molar ratio of cysteine:IL-17 antibodies or
in the reducing mixture is about 66:1.
10. The method according to any one of the preceding claims, wherein the kLa* in the system
during step b) is < about 0.27 h-1, preferably < about 0.18 h-1
.
11. The method according to any one of the preceding claims, wherein prior to step a) the
preparation comprises about 4 mg/ml to about 19.4 mg/ml, e.g., about 10 mg/ml to about 19.4
mg/ml, e.g., about 10 mg/ml to about 15.4 mg/ml, e.g., about 12 mg/ml to about 15 mg/ml, e.g.,
about 13.5 mg/ml of the IL-17 antibodies.
12. The method according to claim 4, wherein the concentration of cysteine in the reducing
mixture is about 4.0 mM to about 8.0 mM, e.g., about 4.8 mM to about 8.0 mM, e.g., about 5.5
mM to about 6.7 mM, e.g., about 6.0 mM.
13. The method according to any one of the preceding claims, wherein prior to step a) the pH
of the preparation is about 7.3 to about 8.5, e.g., about 7.8 to about 8.2, e.g., about 7.9 to about
8.1, e.g., about 8.0.
104
14. The method according to any one of the preceding claims, wherein prior to step a) the
initial percent oxygen saturation in the preparation is at least about 80% or at least about 60%, as
measured using an oxygen probe calibrated at 25oC.
15. The method according to any one of the preceding claims, wherein the reducing mixture
further comprises EDTA, and wherein the concentration of EDTA in the reducing mixture is
about 1.3 mM to about 0.8 mM, e.g., about 1.1 to about 0.9 mM, e.g., about 1.0 mM.
16. The method according to any one of the preceding claims, wherein between step a) and
step b), the reducing mixture is heated to a temperature between about 32oC to about 42oC, e.g.,
to between about 35oC to about 39oC, e.g., to about 37oC .
17. The method according to claim 16, wherein the reducing mixture is heated for about 45 to
about 90 minutes, e.g., about 45 to about 75 minutes, e.g., about 60 minutes.
18. The method according to claim 16, wherein the kLa* in the system during heating is <
about 0.69 h-1, said kLa* being calculated by adapting a saturation curve to a dissolved oxygen
curve.
19. The method according to any one of the preceding claims, wherein the reducing mixture is
incubated according to step b) at a temperature between about 20oC to about 42oC, e.g., about
32oC to about 42oC, e.g., about 35oC to about 39oC, e.g., about 37oC.
20. The method according to claim 7, wherein the reducing mixture is incubated according to
step b) for about 210 to about 420 minutes, e.g., about 210 to about 330 minutes, e.g., about 240
to about 300 minutes, e.g., about 250 minutes.
21. The method according to any one of the preceding claims, wherein during step b) the
reducing mixture is stirred for < about 15 minutes per hour, e.g., < about 5 minutes per hour,
e.g., < about 2 minutes per hour.
105
22. The method according to any one of the preceding claims, wherein the level of intact IL-
17 antibodies in the reducing mixture following step a) decreases to at least about 80% as
measured by sodium dodecyl sulfate capillary electrophoresis (CE-SDS).
23. The method according to any one of the preceding claims, further comprising:
c) cooling the mixture resultant from step b) to room temperature, e.g., between
about 16 oC to about 28 oC.
24. The method according to claim 23, wherein the mixture is cooled according to step c) for
about 45 to about 90 minutes, e.g., about 45 to about 75 minutes, e.g., about 60 minutes.
25. The method according to claim 23, wherein the kLa* in the system during step c) is < 0.69
h-1, said kLa* being calculated by adapting a dissolved oxygen curve to a saturation curve.
26. The method according to claim 23, further comprising:
d) adjusting the pH of the mixture resultant from step c) to between about 5.1 to
about 5.3, e.g., about 5.2.
27. The method according to claim 26, wherein adjusting step d) comprises adding ophosphoric
acid to the mixture resultant from step c).
28. The method according to claim 26, wherein the level of intact IL-17 antibodies in the
mixture resultant from step d) is at least about 90%, as measured by CE-SDS.
29. The method according to claim 26, wherein the level of activity of the IL-17 antibodies in
the mixture resultant from step d) is at least about 90%, as measured by cystamine-cation
exchange chromatography (cystamine-CEX).
30. The method according to any one of the above claims, wherein an oxidized form of the
reducing reagent, e.g., cystine or cystamine, is not added to the reducing mixture.
106
31. The method according to any one of the above claims, wherein a denaturant, e.g.,
guanidine hydrochloride, is not added to the reducing mixture.
32. A method for selectively reducing CysL97 in a preparation of IL-17 antibodies that have
been recombinantly produced by mammalian cells, comprising:
a) contacting the preparation with a set of oxidation/reduction reagents selected from
cysteine/cystine and cysteine/cystamine to form a reducing mixture; and
b) incubating the reducing mixture at a temperature of about 37 °C under anaerobic
conditions for at least about 4 hours, or incubating the reducing mixture at a temperature of about
18-24 °C for about 16-24 hours;
wherein the IL-17 antibodies each comprise an immunoglobulin heavy chain variable
domain (VH) comprising the three complementarity determining regions (CDRs) of the VH set
forth as SEQ ID NO:8 and an immunoglobulin light chain variable domain (VL) comprising the
three CDRs of the VL set forth as SEQ ID NO:10.
33. The method according to claim 32, wherein the molar ratio of oxidation/reduction
reagents in the reducing mixture is between about 4:1 to about 80:1.
34. The method according to claim 33, wherein the molar ratio of oxidation/reduction
reagents in the reducing mixture is between about 26:1 to about 80:1.
35. The method according to claim 33, wherein the set of oxidation/reduction reagents is
cysteine/cystine.
36. The method according to claim 33, wherein the set of oxidation/reduction reagents is
cysteine/cystamine.
37. The method according to claim 32, wherein the molar ratio of cysteine:IL-17 antibodies
in the reducing mixture is between about 21:1 to about 296:1 .
38. The method according to any one of the above claims, wherein the three CDRs of the VH
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set forth as SEQ ID NO:8 are, in sequence, the amino acids sequences set forth as SEQ ID NOs:
1, 2, and 3 or the amino acids sequences set forth as SEQ ID NOs: 11, 12, and 13 and wherein
the three CDRs of the VL set forth as SEQ ID NO:10 are, in sequence, the amino acids sequences
set forth as SEQ ID NOs: 4, 5 and 6.
39. The method according to any one of the above claims, wherein the IL-17 antibodies each
comprise:
i) the VL set forth as SEQ ID NO:10 and the VH set forth as SEQ ID NO:8; or
ii) the full length light chain set forth as SEQ ID NO:14 and the full length heavy
chain set forth as SEQ ID NO:15, with or without the C-terminal lysine.
40. The method according to any one of the above claims, wherein the IL-17 antibodies are
human antibodies of the IgG1 isotype.
41. The method according to any one of the above claims, wherein the IL-17 antibodies are
secukinumab antibodies.
42. The method according to any one of the above claims, wherein the level of activity of the
IL-17 antibodies in the preparation increases by at least about 10 percentage points, at least about
15 percentage points, at least about 20 percentage points, at least about 25 percentage points, or
at least about 30 percentage points within about 60 minutes following step b), as measured by
cystamine-CEX.
43. A method for selectively reducing CysL97 in a preparation of secukinumab antibodies that
have been recombinantly produced by mammalian cells, comprising:
g) adjusting the concentration of secukinumab in the preparation to between
about 4 mg/ml – about 19.4 mg/ml, e.g., about 10 mg/ml - about 19.4 mg/ml, e.g., about 10 -
about 15.4, e.g., about 12 mg/ml - about 15 mg/ml, e.g., about 13.5 mg/ml;
h) adjusting the percent oxygen saturation in the preparation to at least about
60%, e.g., at least about 80%;
i) adjusting the pH of the preparation to about 7.4 - about 8.5, e.g., about
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7.8 - about 8.2, e.g., about 7.9 - about 8.1, e.g., about 8.0;
j) contacting the preparation with cysteine in a vessel to form a reducing
mixture, wherein the concentration of cysteine in the reducing mixture is about 4.0 mM - about
8.0 mM, e.g., about 4.8 mM - about 8.0 mM, e.g., about 5.5 mM - about 6.7 mM, e.g., about 6.0
mM;
k) heating the reducing mixture to a temperature between about 32oC - about
42oC, e.g., to between about 35oC - about 39oC, e.g., to about 37oC, said heating occurring for
about 45 - about 90 minutes, e.g., about 45 - about 75 minutes, e.g., about 60 minutes;
l) incubating the reducing mixture from step e) at a temperature between
about 20oC - about 42oC, e.g., 32oC - about 42oC, e.g., to between about 35oC - about 39oC, e.g.,
to about 37oC, said incubating occurring for about 210 - about 420 minutes, e.g., about 210 -
about 330 minutes, e.g., about 240 - about 300 minutes, e.g., about 250 minutes while
maintaining a volumetric oxygen mass-transfer coefficient (kLa*) in the vessel of < 0.37 h-1, said
kLa* being calculated by adapting a saturation curve to a dissolved oxygen curve,
g) cooling the mixture resultant from step f) to a temperature between about
16oC - about 28oC, said cooling occurring for about 45 - about 90 minutes, e.g., about 45 - about
75 minutes, e.g., about 60 minutes; and
h) adjusting the pH of the mixture resultant from step g) to between about 5.1
- about 5.3, e.g., about 5.2.
44. A purified preparation of IL-17 antibodies made by any one of the preceding claims.
45. The purified preparation according to claim 44, wherein the IL-17 antibodies are
secukinumab antibodies.
46. A purified preparation of secukinumab, wherein the level of intact secukinumab in the
preparation is at least about 90%, as measured by sodium dodecyl sulfate capillary
electrophoresis (CE-SDS), and wherein the level of activity of secukinumab in the preparation is
at least about 90%, as measured by cystamine-CEX.