NZ749400A - Production of activated clostridial neurotoxins - Google Patents
Production of activated clostridial neurotoxinsInfo
- Publication number
- NZ749400A NZ749400A NZ749400A NZ74940017A NZ749400A NZ 749400 A NZ749400 A NZ 749400A NZ 749400 A NZ749400 A NZ 749400A NZ 74940017 A NZ74940017 A NZ 74940017A NZ 749400 A NZ749400 A NZ 749400A
- Authority
- NZ
- New Zealand
- Prior art keywords
- neurotoxin
- clostridial neurotoxin
- bont
- disorders
- clostridial
- Prior art date
Links
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Abstract
The present invention relates to a method of producing activated clostridial neurotoxins that are essentially free of unactivated products, to compositions comprising such and to their use in therapy.
Description
TION OF ACTIVATED CLOSTRIDIAL NEUROTOXINS
FIELD OF THE INVENTION
The present invention relates to a method of producing activated clostridial
neurotoxins that are essentially free of unactivated products, to compositions
comprising such and to their use in therapy.
BACKGROUND
Bacteria in the genus idia produce highly potent and ic protein
toxins, which can poison neurons and other cells to which they are delivered.
Examples of such clostridial toxins include the neurotoxins produced by C.
tetani (TeNT) and by C. botulinum (BoNT) serotypes A-G, as well as those
ed by C. baratii and C. butyricum.
Among the clostridial neurotoxins are some of the most potent toxins known.
By way of e, botulinum neurotoxins have median lethal dose (LD50)
values for mice ranging from 0.5 to 5 ng/kg, depending on the serotype. Both
s and botulinum toxins act by inhibiting the function of affected neurons,
specifically the release of neurotransmitters. While num toxin acts at the
neuromuscularjunction and inhibits cholinergic transmission in the peripheral
nervous system, s toxin acts in the central nervous system.
In nature, clostridial neurotoxins are synthesised as a single-chain polypeptide
that is modified post-translationally by a proteolytic ge event to form two
polypeptide chains joined together by a hide bond. Cleavage occurs at
a specific cleavage site, often referred to as the activation site that is located
between the cysteine residues that provide the inter-chain disulphide bond. It
is this di-chain form that is the most active form of the oxin. The two
chains are termed the heavy chain (H-chain), which has a molecular mass of
approximately 100 kDa, and the light chain (L-chain), which has a molecular
mass of approximately 50 kDa. The H-chain comprises an N-terminal
translocation component (HN domain) and a C-terminal targeting component
(Hc domain). The ge site is located between the L-chain and the
translocation domain components. Following g of the Hc domain to its
target neuron and internalisation of the bound toxin into the cell via an
endosome, the HN domain translocates the L—chain across the endosomal
membrane and into the cytosol, and the L-chain provides a protease function
(also known as a non-cytotoxic protease).
Non-cytotoxic proteases act by proteolytically ng intracellular transport
proteins known as SNARE proteins (e.g. SNAP-25, VAMP, or Syntaxin) — see
Gerald K (2002) "Cell and Molecular Biology” (4th edition) John Wiley & Sons,
Inc. The acronym SNARE derives from the term Soluble NSF Attachment
Eceptor, where NSF means N—ethylmaleimide-Sensitive Eactor. SNARE
proteins are integral to intracellular e fusion, and thus to secretion of
molecules via vesicle transport from a cell. The protease function is a zinc-
dependent endopeptidase ty and exhibits a high substrate specificity for
SNARE proteins. Accordingly, once delivered to a desired target cell, the non-
cytotoxic protease is capable of inhibiting cellular secretion from the target cell.
The L-chain proteases of clostridial neurotoxins are non-cytotoxic ses
that cleave SNARE ns.
In view of the tous nature of SNARE proteins, clostridial neurotoxins
such as botulinum toxin have been successfully employed in a wide range of
therapies.
By way of example, we refer to William J. Lipham, Cosmetic and Clinical
Applications of Botulinum Toxin (Slack, Inc., 2004), which describes the use of
idial oxins, such as botulinum neurotoxins (BoNTs), ,
BoNT/B, BoNT/C1, , BoNT/E, BoNT/F and BoNT/G, and tetanus
neurotoxin (TeNT), to inhibit neuronal transmission in a number of therapeutic
and cosmetic or aesthetic applications - for example, marketed botulinum toxin
products are currently approved as therapeutics for indications including focal
spasticity, upper limb spasticity, lower limb spasticity, cervical dystonia,
blepharospasm, hemifacial spasm, hyperhidrosis of the axillae, chronic
migraine, neurogenic detrusor overactivity, glabellar lines, and severe l
canthal lines. In addition, clostridial oxin therapies are described for
treating neuromuscular disorders (see US 6,872,397); for treating uterine
disorders (see US 2004/0175399); for treating ulcers and gastroesophageal
reflux disease (see US 086531); for treating dystonia (see US
6,319,505); for treating eye disorders (see US 2004/0234532); for treating
blepharospasm (see US 2004/0151740); for treating smus (see US
2004/0126396); for treating pain (see US 6,869,610, US 6,641,820, US
6,464,986, and US 6,113,915); for treating fibromyalgia (see US 6,623,742,
US 2004/0062776); for treating lower back pain (see US 2004/0037852); for
treating muscle injuries (see US 319); for treating sinus headache (see
US 6,838,434); for treating n headache (see US 6,776,992); for ng
headache (see US 6,458,365); for reduction of migraine headache pain (see
US 5,714,469); for ng vascular diseases (see US 6,767,544); for
treating neurological disorders such as Parkinson's disease (see US
6,620,415, US 6,306,403); for treating neuropsychiatric disorders (see US
2004/0180061, US 2003/0211121); for treating endocrine disorders (see US
6,827,931); for treating thyroid disorders (see US 6,740,321); for treating
cholinergic influenced sweat gland ers (see US 6,683,049); for treating
diabetes (see US 075, US 6,416,765); for treating a pancreatic disorder
(see US 6,261,572, US 306); for treating cancers such as bone tumors
(see US 6,565,870, US 605, US 6,139,845, US 2005/0031648); for
treating otic disorders (see US 6,358,926, US 6,265,379); for treating
autonomic disorders such as gastrointestinal muscle disorders and other
smooth muscle dysfunction (see US 5,437,291); for treatment of skin lesions
associated with cutaneous cell-proliferative disorders (see US 5,670,484); for
management of neurogenic inflammatory disorders (see US 6,063,768); for
reducing hair loss and stimulating hair growth (see US 6,299,893); for treating
downturned mouth (see US 6,358,917); for reducing appetite (see US
2004/40253274); for dental therapies and procedures (see US 2004/0115139);
for treating neuromuscular disorders and conditions (see US 2002/0010138);
for treating various disorders and conditions and ated pain (see US
013692); for treating conditions ing from mucus hypersecretion
such as asthma and COPD (see WO 00/10598); and for treating non-neuronal
conditions such as inflammation, endocrine ions, exocrine conditions,
2017/066361
immunological conditions, cardiovascular conditions, bone conditions (see WO
01/21213). All of the above publications are hereby incorporated by reference
in their entirety.
The use of totoxic proteases such as clostridial neurotoxins (e.g. BoNTs
and TeNT) in therapeutic and cosmetic treatments of humans and other
mammals is anticipated to expand to an ever-widening range of diseases and
ailments that can benefit from the properties of these toxins.
Traditionally, production of clostridial neurotoxins is carried out by culture of C.
num bacteria, followed by isolation and purification of the clostridial
neurotoxin complex. However, C. botu/inum are forming bacteria and
therefore e list culture equipment and facilities, which are not
required for the culture of bacteria such as Escherichia coli (E. coli). The
increasing use of clostridial neurotoxins has therefore led to a need for
alternative and/or improved methods for producing and purifying idial
neurotoxins.
Clostridial neurotoxins are initially expressed as single chain polypeptides. In
order to be fully active, the single chain form must be converted into a di-chain
form, which requires proteolytic cleavage at a site located between the light
and heavy chains (“activation loop”). In vitro activation of clostridial
neurotoxins can be achieved by the addition of a suitable protease. It is
however a recurrent issue in the art that unwanted proteolytic activity
frequently occurs at sites outside the activation loop before full activation is
achieved, resulting in the formation of undesirable ated” (or
“overactivated”) products. The formation of such products is particularly
problematic when the activated di-chain clostridial neurotoxins are ed for
use as pharmaceutical products as a highly pure and functional preparation is
hed solutions to the problem require some form of sequence
modification of the BoNT amino acid ce; examples include the insertion
of specific se recognition sites within the activation loop, thereby
changing its sequence.
2017/066361
For example, W00114570 describes recombinant nucleic acid molecules
encoding BoNT proteins. However, the nucleic acid molecules of W001 14570
are ed to replace the native cleavage site with a tive ge
site. Thus, the method of W00114570 also teaches that insertion of a non-
native cleavage site is required for optimal BoNT expression.
U820080103098 describes a method for producing recombinant BoNT
proteins in a di-chain form comprising expression of a recombinant nucleic acid
construct in an E. coli host cell. r, said method requires the insertion
of a specific, non-native (i.e. non—clostridial) pentapeptide sequence into a loop
domain of the neurotoxin. The inserted pentapeptide sequence forms an
activation ge site that is cleaved by an nous E. coli protease
upon cell lysis. The method of U820080103098 therefore teaches that in order
to achieve l BoNT expression, the BoNT sequence must be ed by
the insertion of a non-native cleavage site.
W02010094463 describes a process for separating processed BoNT from
unprocessed or partially processed BoNT requiring the use of antibodies
against the proteolytically unprocessed and/or partially processed BoNT.
Another approach described in W02012020057 is based on the insertion of a
modified linker conferring a physicochemical property between the light chain
and the heavy chain flanked by protease cleavage sites. The linker’s
physicochemical property is such as it must allow for separation of partially
processed or unprocessed BoNT from processed (activated) BoNT.
There is therefore a need in the art for methods for producing essentially pure,
biologically active preparations of full-length activated clostridial neurotoxin not
relying on the insertion of exogenous sequences. The present ion solves
this problem by providing a novel method as specified in the claims that avoids
the requirement to modify the idial neurotoxin amino acid sequence or
use purification tags.
WO 02348 2017/066361
SUMMARY OF THE INVENTION
According to a first aspect the present invention provides a method for
producing an activated clostridial neurotoxin, sing contacting a single
chain idial neurotoxin with an activation enzyme until at least 90% of the
single chain clostridial neurotoxin polypeptide is ted into a di-chain form.
In a second aspect the invention provides an active di-chain clostridial
neurotoxin ed by the method according to the invention.
In a third aspect the invention provides a pharmaceutical ition
comprising an active di-chain clostridial neurotoxin according to the invention
which is essentially free of single chain idial neurotoxin.
DETAILED DESCRIPTION OF THE INVENTION
This invention relates to a method for the manufacture of biologically active,
full-length, essentially pure preparations of clostridial neurotoxins by a counter-
intuitive approach.
The intuitive solution to the problem of producing essentially pure preparations
of clostridial neurotoxins is to adjust the conditions of the activation process so
that a minimum amount of truncated product is formed. However, the inventors
have found that using such an approach results in a proportion of the clostridial
neurotoxin remaining uncleaved (unactivated). In other words, such a process
yields a mixture of full-length activated, full-length unactivated (single chain)
and truncated clostridial neurotoxin products. In this t, it is important to
note that full-length unactivated and truncated activated products are
undesirable, even more so when the activated di-chain clostridial neurotoxins
are intended for use as ceutical products. Such undesirable products
must therefore be removed during the subsequent steps of the manufacturing
The inventors have further found that it is extremely hard to te full-length
ted clostridial neurotoxin (di—chain) from full-length unactivated clostridial
neurotoxin (single chain).
The inventors have surprisingly found that by allowing the activation step to
progress to a later stage at which essentially no full-length unactivated product
remains, renders it easier to remove the unwanted by-products (over-activated
or truncated products) in subsequent purification steps.
Without willing to be bound by theory, it is hypothesized that the difficulties
encountered with respect to separating full-length activated clostridial
botulinum toxin from full-length vated botulinum toxin are due to
insufficient exploitable physicochemical differences between full-length
activated botulinum toxin and full-length unactivated botulinum toxin. It is
further hypothesized that unwanted ted clostridial neurotoxin by-
products have greater physicochemical differences compared to activated full-
length idial neurotoxins due to the change in primary amino acid
ce between the ts. These greater physicochemical differences
can be exploited by purification techniques known in the art, for example
column chromatography, to achieve separation of full-length activated
clostridial neurotoxin and obtain an essentially pure product le for use in
therapy.
Therefore, in a first aspect, there is provided a method for producing an
ted clostridial neurotoxin, comprising contacting a single chain clostridial
neurotoxin with an tion enzyme until over 90% of the single chain
idial neurotoxin polypeptide is converted into a di-chain form.
Preferably, the single chain clostridial neurotoxin is contacted with the
activation enzyme until over 95% of the single chain clostridial neurotoxin
polypeptide is converted into a di-chain form. More preferably, the single chain
idial neurotoxin is contacted with the activation enzyme until over 96%,
97%, 98%, 99%, 99,5%, 999% or 100% of the single chain clostridial
neurotoxin polypeptide is converted into a di-chain form.
The term “active clostridial neurotoxin” refers herein to a idial neurotoxin
that is capable of binding to a target cell, getting internalised into said cell and
of inhibiting SNARE-driven secretion of neurotransmitters from said cell. It is
well known in the art that the level of biological activity of a clostridial
neurotoxin is much higher when the idial neurotoxin is in a di-chain (or
“activated”) form than when it is in a single chain form. A biologically active
clostridial neurotoxin is therefore ably a di-chain (or “‘activated”)
clostridial neurotoxin.
The term “activation” refers herein to the conversion of a single chain clostridial
neurotoxin polypeptide into a di-chain (or active) form.
An “activation enzyme” (or “activation se”) as used herein means an
endopeptidase which is capable of cleaving a single chain clostridial
neurotoxin at a site located between the clostridial oxin light chain and
heavy chain (referred to in the art as the “activation loop”), such as to allow for
the ion of a fully active di—chain clostridial oxin (or “activated
idial neurotoxin”).
Examples of activation s suitable for the present invention include
members of the cysteine, serine and metalloprotease families such as trypsin,
Lys-C, Lys-N and arginyl endopeptidases (endoproteinase Arg-C, LeR), as
well as an active BoNT hydrolase as sed in EP 2 524 963 A1, hereby
incorporated by reference in its entirety.
In a preferred embodiment the activation enzyme is a trypsin.
In a most red embodiment the activation enzyme is a bovine trypsin.
The inventors indeed found that a bovine trypsin is more suitable for activating
a botulinum neurotoxin than other sources of trypsin, such as porcine trypsin.
Indeed, the inventors found that with bovine trypsin full activation can be
achieved with the occurrence of very little undesirable truncated products as
compared to when porcine trypsin is used.
This came as a surprising finding to the inventors in view of the fact that porcine
trypsin amino acid sequence is the most commonly used recombinant GMP-
grade trypsin sequence and appeared to be the obvious trypsin source for this
purpose. It is indeed ble to use GMP grade, animal-free raw materials
for the manufacture of ade idial neurotoxins which are destined
to be used in therapy.
In a preferred embodiment, the activation enzyme is a GMP-grade enzyme
such as a GMP-grade trypsin, more preferably a GMP-grade bovine trypsin,
more preferably still a GMP-grade recombinant bovine trypsin. “GMP-grade”
means manufactured to y standards and with traceability suitable for use
in GMP (Good Manufacturing Practices) manufacture.
In a preferred embodiment, the activation enzyme is a bovine n such as
a native trypsin obtained from bovine pancreas (TPCK-treated or non-TPCK
treated) or a inant bovine trypsin.
According to the invention, a bovine trypsin is a protein having at least 90%,
for example at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or
100% identity with SEQ ID NO: 1 (UniProtKB Accession Number P00760).
A bovine trypsin may be obtainable from any suitable source and is
commercially available eg. from d Biotechnology ute (TrypZean®).
Alternatively, a bovine trypsin may be obtained by recombinant expression of
an amino acid sequence encoding a protein having at least 90% identity with
SEQ ID NO: 1.
The inventors also found that the specificity of bovine trypsin for the activation
loop of BoNT/E is higher at a Ph around 6-7. This result was unexpected as
the performance of trypsin is widely known in the art to be optimal at about pH
In a preferred embodiment, the step of contacting the single chain clostridial
neurotoxin with a bovine trypsin is med at a pH of between 5 and 7,5,
preferably between 6 and 7, for example at a pH of approximatively 6,5.
The step of contacting the single chain clostridial neurotoxin with the activation
enzyme, preferably trypsin, is preferably carried out for a duration of 10 to 50
hours, preferably between 15 and 30 hours, for example about 15, 16, 17, 18,
19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 hours.
The step of ting the single chain clostridial neurotoxin with trypsin is
preferably at room temperature (or t temperature). Room temperature
is typically between 16 and 27 °C, preferably between 18 and 25 ° C, for
example about 18, 19, 20, 21, 22,23, 24 or 25 °C.
The tration of trypsin is typically between 0.5 and 50 pg per mg of
clostridial neurotoxin, ably between 1 and 20 pg per mg of clostridial
neurotoxin, more preferably between 2 and 10 pg per mg of clostridial
neurotoxin, for example about 2, 3, 4, 5, 6, 7, 8, 9 or 10 ug of n per mg
of clostridial neurotoxin.
The concentration of trypsin can be expressed in USP (United States
Pharmacopeia) units, in which case it is typically between 1 and 500 USP units
per mg of clostridial neurotoxin, preferably between 3 and 100 USP units per
mg of clostridial neurotoxin, more preferably n 5 and 50 USP units per
mg of clostridial neurotoxin, for e about 5, 7.5, 10, 12.5, 15, 20, 25, 30,
, 40, 45 or 50 USP units of trypsin per mg of clostridial neurotoxin.
According to a preferred embodiment, the step of contacting the single chain
idial neurotoxin with trypsin is carried out at room temperature at a pH
between 6 and 7, with a bovine trypsin at a concentration between 1 and 20
pg per mg of idial neurotoxin for a duration of 15 to 30 hours.
According to another preferred embodiment, the step of ting the single
chain clostridial neurotoxin with trypsin is carried out at room temperature at a
pH between 6 and 7, with a bovine trypsin at a concentration between 3 and
100 USP units per mg of clostridial neurotoxin for a duration of 15 to 30 hours.
According to an embodiment of the t invention, the method further
comprises a step of removing truncated (overactivated) clostridial neurotoxin,
in particular truncated di-chain clostridial neurotoxin.
The term “truncated di-chain clostridial neurotoxin” (or “unwanted by—product”
or “overactivated clostridial neurotoxin”) refers to a product resulting from
cleavage of a single chain botulinum neurotoxin at more than one site and l or
at a site outside the activation loop.
Such a step can be performed for e by contacting the obtained di-chain
clostridial neurotoxin with a suitable chromatography resin. An e of a
le tography resin is a mixed mode chromatography resin. An
example of a suitable mixed mode chromatography resin is a Ceramic
Hydroxyapatite (CHT) Type II 40 micron resin (BioRad).
Many different types of clostridial neurotoxins are suitable for use in the
present ion. Thus, in the context of the present invention, the term
“clostridial neurotoxin” (or “clostridial toxin”) embraces toxins produced by C.
botulinum inum neurotoxin serotypes A, B, C1, D, E, F and G), C. tetani
(tetanus neurotoxin), C. butyricum inum neurotoxin serotype E), and C.
baratii (botulinum neurotoxin serotype F), as well as modified clostridial
neurotoxins or derivatives derived from any of the foregoing. The term
“clostridial neurotoxin” also embraces lly occurring botulinum neurotoxin
hybrids, mosaics and chimera.
Therefore in one embodiment a clostridial neurotoxin of, or for use in the
present invention may be obtainable from one or more Clostridia selected from
the group consisting of: Clostridia botulinum, Clostridia tetani, Clostridia baratii
and C. butyricum.
Botulinum neurotoxin (BoNT) is produced by C. botulinum in the form ofa large
protein complex, consisting of BoNT itself complexed to a number ofaccessory
proteins. There are at t seven different classes of botulinum neurotoxin,
namely: botulinum neurotoxin serotypes A, B, C1, D, E, F and G, all of which
share similar structures and modes of . Different BoNT serotypes can be
distinguished based on inactivation by specific neutralising era, with such
classification by serotype correlating with tage sequence identity at the
amino acid level. BoNT proteins of a given serotype are further divided into
different subtypes on the basis of amino acid percentage sequence identity.
Suitably the clostridial neurotoxin of, or for use in, the present invention may
be a botulinum oxin (BoNT), ably one or more BoNT(s) ed
from the group consisting of: BoNT/A, BoNT/B, BoNT/C1, BoNT/D, BoNT/E,
BoNT/F and BoNT/G.
In one embodiment the clostridial neurotoxin may be BoNT/A. A reference
BoNT/A sequence has the UniProtKB Accession Number P10845 (SEQ ID
NO: 2).
In another embodiment the idial neurotoxin may be BoNT/B. A reference
BoNT/B sequence has the tKB Accession Number P10844 (SEQ ID
NO: 3).
WO 02348
In another ment the clostridial neurotoxin may be BoNT/C. A reference
BoNT/Ci sequence has the UniProtKB Accession Number P18640 (SEQ ID
NO: 4).
In another embodiment the idial neurotoxin may be BoNT/D. A reference
BoNT/D ce has the UniProtKB Accession Number P19321 (SEQ ID
NO: 5).
In another embodiment the clostridial neurotoxin may be BoNT/E. A reference
BoNT/E sequence has the UniParc I.D UPI00000010A3 (SEQ ID NO: 6).
In another embodiment the clostridial neurotoxin may be BoNT/F. A reference
BoNT/F ce has the UniProtKB Accession Number YP_001390123
(SEQ ID NO: 7).
In another embodiment the clostridial neurotoxin may be BoNT/G. A reference
BoNT/G sequence has the UniProtKB Accession Number Q60393 (SEQ ID
NO: 8).
In one ment the clostridial neurotoxin may be a TeNT. A reference
TeNT ce has the UniProtKB Accession Number P04958 (SEQ ID NO:
In one embodiment the clostridial neurotoxin of, or for use in, the present
ion comprises a BoNT/E activation loop. A BONT/E activation loop may
be defined as comprising SEQ ID NO: 10: “CKNIVSVKGIRKSIC”
(corresponding to amino acid residues C412 to C426 of SEQ ID NO: 6), or a
sequence differing from SEQ ID NO: 10 by 1, 2, 3, 4 or 5 amino acid residue
insertions, deletions or substitutions.
In one embodiment, a BONT/E activation loop has a sequence consisting of
SEQ ID NO: 10. In another embodiment, a BONT/E activation loop has a
sequence differing from SEQ ID NO: 10 by 1amino acid residue insertion,
deletion or substitution. In another embodiment, a BONT/E activation loop has
a sequence differing from SEQ ID NO: 10 by 2 amino acid residue insertions,
deletions or substitutions. In another embodiment, a BONT/E tion loop
has a sequence differing from SEQ ID NO: 10 by 3 amino acid residue
ions, deletions or substitutions. In another embodiment, a BONT/E
activation loop has a sequence differing from SEQ ID NO: 10 by 4 amino acid
residue insertions, deletions or substitutions. In another embodiment, a
BONT/E activation loop has a sequence differing from SEQ ID NO: 10 by 5
amino acid residue insertions, deletions or substitutions.
In a preferred embodiment, the clostridial neurotoxin is a BoNT/E, for example
a BoNT/E having an amino acid sequence at least 90%, 91%, 92%, 93%, 94%,
95%, 96%, 97%, 98%, 99% or 100% identical to SEQ ID NO: 6.
The term “clostridial neurotoxin” is also intended to embrace modified
clostridial neurotoxins and derivatives thereof, including but not limited to those
described below. A modified clostridial neurotoxin or tive may contain
one or more amino acids that has been modified as compared to the native
(unmodified) form of the clostridial neurotoxin, or may contain one or more
ed amino acids that are not present in the native (unmodified) form of the
clostridial neurotoxin, or may contain one or more deleted amino acids as
compared to the native (unmodified) form of the clostridial neurotoxin. By way
of example, a modified clostridial neurotoxin may have modified amino acid
sequences in one or more domains ve to the native (unmodified)
clostridial neurotoxin sequence. Such cations may modify functional
aspects of the neurotoxin, for example biological activity or persistence.
Modified clostridial oxins may have one or more modifications in the
amino acid sequence of the heavy chain (such as a modified Hc ),
wherein said modified heavy chain binds to target nerve cells with a higher or
lower ty than the native (unmodified) clostridial neurotoxin. Such
modifications in the Hc domain can include modifying residues in the
ganglioside g site of the Hc domain or in the protein (SV2 or
synaptotagmin) binding site that alter binding to the ganglioside receptor
and/or the n receptor of the target nerve cell. Examples of such ed
clostridial neurotoxins are described in WO 2006/027207 and WO
2006/114308, both of which are hereby orated by reference in their
entirety.
A modified clostridial neurotoxin may have one or more modifications in the
amino acid sequence of the light chain, for example modifications in the
2017/066361
substrate binding or catalytic domain which may alter or modify the SNARE
protein specificity of the modified LC. Examples of such modified clostridial
neurotoxins are described in and US 2011/0318385, both of
which are hereby incorporated by reference in their entirety.
A modified clostridial neurotoxin may comprise one or more modifications that
increases or decreases the biological ty and/or the biological persistence
of the ed clostridial neurotoxin.
The term “clostridial neurotoxin” is ed to embrace chimeric (or hybrid)
clostridial neurotoxins. A ic clostridial neurotoxin comprises at least a
portion of a light chain from one clostridial neurotoxin type or subtype thereof,
and at least a portion of a heavy chain from another clostridial neurotoxin type
or subtype.
In one embodiment the chimeric idial neurotoxin may contain the entire
light chain from one clostridial oxin type or subtype and the heavy chain
from another clostridial neurotoxin type or subtype. In another embodiment, a
chimeric clostridial neurotoxin may contain a portion (e.g. the binding domain)
of the heavy chain of one clostridial neurotoxin type or subtype, with r
portion of the heavy chain being from another idial neurotoxin type or
subtype. Similarly or alternatively, the therapeutic element may comprise light
chain portions from different clostridial neurotoxin types or subtypes. Such
hybrid or chimeric clostridial neurotoxins are useful, for example, as a means
of delivering the therapeutic benefits of such idial neurotoxins to patients
who are immunologically resistant to a given clostridial oxin subtype, to
patients who may have a lower than average concentration of receptors to a
given clostridial neurotoxin heavy chain g domain, or to ts who
may have a protease-resistant variant of the membrane or vesicle toxin
substrate (e.g., SNAP-25, VAMP and syntaxin). Hybrid and chimeric clostridial
neurotoxins are described in US 8,071,110 and in GB1607901.4 (not yet
published), which are hereby orated by reference in their entirety. Thus,
in one embodiment, the clostridial neurotoxin for purification according to a
method or use of the present invention may be an engineered clostridial
neurotoxin, suitably it may be an ered chimeric clostridial neurotoxin.
The term “clostridial neurotoxin” is intended to embrace geted clostridial
neurotoxins. In a re-targeted clostridial neurotoxin, the clostridial neurotoxin is
ed to include an exogenous ligand known as a Targeting Moiety (TM).
The TM is selected to provide binding specificity for a desired target cell, and
as part of the re-targeting s the native binding portion of the clostridial
neurotoxin (eg. the Hc domain, or the H00 ) may be removed. Re-
targeting technology is described, for example, in: EP-B-0689459; WO
1994/021300; EP-B-0939818; US 6,461,617; US 596; WO
1998/007864; 826051; US 5,989,545; US 6,395,513; US 6,962,703;
; EP-B-0996468; US 7,052,702; ; EP-B-
1107794; US 6,632,440; WO 2000/010598; WO 2001/21213; WO
2006/059093; ; ; ; WO
1192; and ; all of which are hereby incorporated by
reference in their ty. Thus, in one embodiment, the engineered clostridial
neurotoxin for use in the present invention may be an engineered re-targeted
idial neurotoxin.
The present invention also embraces the use of clostridial oxins
comprising a “destructive cleavage site”. In said clostridial neurotoxins, a nonnative
protease cleavage site is incorporated into the clostridial neurotoxin, at
a location chosen such that cleavage at said site will decrease the activity of,
or inactivate, the clostridial neurotoxin. The destructive protease cleavage site
can be susceptible to ge by a local protease, in the event that the
clostridial neurotoxin, following administration, migrates to a non-target
location. Suitable non—native protease cleavage sites include those described
above. Clostridial neurotoxins comprising a destructive cleavage site are
described in and , both of which are
hereby incorporated by reference in their entirety.
In a preferred ment the clostridial neurotoxin of the present invention
or for use in the present ion is free from the complexing proteins that are
present in a naturally occurring clostridial neurotoxin complex.
In one embodiment a clostridial neurotoxin of, or for use in, the present
invention may comprise a ptide sequence shown as SEQ ID NO: 2, 3,
4, 5, 6, 7, 8 or 9 or a polypeptide sequence having at least 65% or 70%
ce identity thereto.
In one embodiment a clostridial neurotoxin of, or for use in, the present
invention may comprise a polypeptide sequence shown as SEQ ID NO: 2, 3,
4, 5, 6, 7, 8 or 9 or a polypeptide sequence having at least 75% or 80%
sequence identity thereto.
In one embodiment a clostridial neurotoxin of, or for use in, the present
ion may comprise a polypeptide sequence shown as SEQ ID NO: 2, 3,
4, 5, 6, 7, 8 or 9 or a polypeptide sequence having at least 85% or 90%
sequence identity thereto.
In one embodiment a idial neurotoxin of, or for use in, the present
invention may comprise a polypeptide sequence shown as SEQ ID NO: 2, 3,
4, 5, 6, 7, 8 or 9 or a polypeptide ce having at least 95% or 99%
sequence identity thereto.
In a preferred embodiment a clostridial neurotoxin of, or for use in, the present
invention comprises a polypeptide sequence shown as SEQ ID NO: 6 or a
polypeptide sequence having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%
or 99% sequence ty thereto.
The "percent sequence ty" between two or more nucleic acid or amino
acid sequences is a function of the number of identical ons shared by the
sequences. Thus, % identity may be calculated as the number of identical
tides / amino acids divided by the total number of nucleotides / amino
acids, multiplied by 100. Calculations of % sequence identity may also take
into account the number of gaps, and the length of each gap that needs to be
introduced to optimize ent of two or more sequences. Sequence
comparisons and the determination of percent identity between two or more
sequences can be d out using specific mathematical algorithms, such as
BLAST, which will be familiar to a skilled person.
In one embodiment, the single chain clostridial neurotoxin for use in the
present invention is ed by expressing a gene encoding the single chain
clostridial neurotoxin in a heterologous host cell, such as a bacterial, insect,
yeast, ial, mammalian or plant cell, or in a cell-free system. Preferably,
the heterologous host cell is E. coli.
In another aspect, the present invention provides an active di-chain clostridial
neurotoxin obtainable by the method according to the invention.
In r , the present invention provides a pharmaceutical
composition comprising an active di-chain clostridial neurotoxin according to
the invention, wherein said composition is essentially free of single chain
clostridial neurotoxin.
The term “essentially pure” or “essentially free of” as used herein means that
the level of undesirable contaminants (or by-products) is lower than 10%, for
example lower than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%. or than 0.1%.
In a preferred embodiment embodiment, the pharmaceutical composition
according to the invention comprises less than 10%, for example lower than
9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or than 0.1% single chain idial
neurotoxin.
In a more preferred one embodiment, the pharmaceutical composition
according to the invention further comprises less than 10%, for e lower
than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or than 0.1% truncated (over-
activated) clostridial neurotoxin.
In a red embodiment, the pharmaceutical composition according to the
invention comprises less than 5% single chain clostridial neurotoxin and less
than 5% truncated di-chain clostridial neurotoxin.
In a more preferred embodiment, the pharmaceutical composition ing
to the invention comprises less than 1% single chain clostridial neurotoxin and
less than 1% truncated di-chain clostridial neurotoxin.
In a more preferred ment, the pharmaceutical composition according
to the invention ses less than 0.1% single chain clostridial oxin
and less than 0.1 % truncated di-chain clostridial neurotoxin.
The relative amounts of single chain, di-chain and truncated di-chain idial
neurotoxin can be assessed by methods well known in the art, for example by
using SDS—PAGE followed by a densitometry analysis to determine relative
amounts (see eg example 1), by capillary electrophoresis or by UPLC (Ultra-
Performance Liquid Chromatography) methodologies for ment of purity
(size exclusion, ion exchange, e-phase, hydrophobic interaction
chromatography).
In one embodiment, the pharmaceutical composition according to the invention
is for use in therapy.
The pharmaceutical composition according to the invention can be employed
for treating or preventing a disease, condition or syndrome selected from
ar disorders, neuromuscular disorders, neurological disorders,
ophtalmological disorders, pain disorders, psychological disorders, articular
disorders, inflammatory disorders, endocrine disorders and urological
disorders, including:
- ophtalmological disorders selected from the group consisting of
blepharospasm, strabismus (including restrictive or myogenic strabismus),
amblyopia, oscillopsia, protective , therapeutic ptosis for l
protection, nystagmus, estropia, ia, entropion, eyelid tion, orbital
myopathy, phoria, concomitant misalignment, nonconcomitant
misalignment, primary or secondary esotropia or pia, internuclear
lmoplegia, skew ion, Duane' s syndrome and upper eyelid
retraction;
- movement disorders including hemifacial spasm, torticollis, spasticity of
the child or of the adult (e.g. in cerebral palsy, post-stroke, multiple sis,
traumatic brain injury or spinal cord injury patients), idiopathic focal dystonias,
muscle stiffness, 's cramp, hand dystonia, VI nerve palsy, oromandibular
dystonia, head tremor, tardive esia, tardive dystonia, occupational
cramps (including ans' cramp) facial nerve palsy, jaw closing spasm,
facial spasm, synkinesia, tremor, primary writing tremor, myoclonus, stiff-
person-syndrome, foot dystonia, facial paralysis, painful-arm-and-moving-
fingers-syndrome, tic disorders, dystonic tics, Tourette' s syndrome,
neuromyotonia, trembling chin, lateral rectus palsy, dystonic foot inversion, jaw
dystonia, Rabbit syndrome, cerebellar tremor, lll nerve palsy, palatal
myoclonus, akasthesia, muscle cramps, IV nerve palsy, freezing-of— gait,
extensor truncal dystonia, post-facial nerve palsy synkinesis, secondary
dystonia, Parkinson's disease, Huntington' s chorea, epilepsy, off period
dystonia, cephalic tetanus, myokymia and benign cramp-fasciculation
- otorhinolaryngological ers including spasmodic dysphonia, otic
disorders, hearing impairment, ear click, us, vertigo, e's e,
cochlear nerve dysfunction, stuttering, cricopharyngeal dysphagia, bruxism,
closure of larynx in chronic aspiration, vocal fold granuloma, ventricular
dystonia, ventricular dysphonia, mutational dysphonia, trismus, snoring, voice
, aspiration, tongue protrusion dystonia, palatal tremor, deep bite of lip
and laryngeal dystonia; First Bite Syndrome;
- gastrointestinal disorders including achalasia, anal fissure,
constipation, temperomandibular joint dysfunction, sphincter of Oddi
dysfunction, sustained sphincter of Oddi hypertension, intestinal muscle
disorders, puborectalis syndrome, s, pyloric spasm, gall bladder
ction, gastrointestinal or oesophageal motility dysfunction, diffuse
ageal spasm and gastroparesis;
- urogenital disorders ing detrusor sphincter dyssynergia, detrusor
hyperreflexia, neurogenic bladder dysfunction (eg in Parkinson's disease,
spinal cord , stroke or multiple sclerosis patients), overactive r,
neurogenic detrusor overactivity, bladder spasms, urinary incontinence,
urinary retention, hypertrophied bladder neck, g dysfunction, interstitial
cystitis, vaginismus, endometriosis, pelvic pain, prostate gland enlargement
n Prostatic Hyperplasia) cancer and priapism;
, prostatodynia, prostate
- dermatological disorders including cutaneous cell proliferative
disorders, skin wounds, psoriasis, rosacea, acne; rare hereditary skin
disorders such as Fox—Fordyce syndrome or Hailey-Hailey disease; keloid and
rophic scar reduction; pore size reduction; inflammatory conditions of
the skin; painful inflammatory conditions of the skin;
- pain disorders including back pain (upper back pain, . lower back pain),
myofascial pain, tension headache, fibromyalgia, painful syndromes, myalgia,
2017/066361
migraine, whiplash, joint pain, post-operative pain, pain not associated with a
muscle spasm and pain associated with smooth muscle disorders;
- inflammatory disorders including pancreatitis, neurogenic inflammatory
disorders (including gout, tendonitis, bursitis, dermatomyositis and ankylosing
spondylitis);
- secretory disorders such as excessive gland secretions, hyperhidrosis
(including axillary hyperhidrosis, palmar hyperhidrosis and Frey' s syndrome),
hypersalivation, sialorrhoea, drosis, mucus hypersecretion,
acrimation, holocrine gland dysfunction; excess sebum secretion;
- respiratory disorders including rhinitis (including allergic rhinitis), COPD,
asthma and tuberculosis;
- hypertrophic disorders including muscle enlargement, masseteric
hypertrophy, acromegaly and neurogenic tibialis or rophy with
myalgia;
- articular ers including tennis elbow (or dilytis of the elbow),
inflammation of joints, coxarthrosis, rthritis, rotator muscle cap
pathology of the shoulder, rheumatoid arthritis and carpal tunnel syndrome;
- endocrine disorders like type 2 diabetes, hyperglucagonism,
hyperinsulinism, hypoinsulinism, hypercalcemia, hypocalcemia, thyroid
disorders (including Grave's disease, thyroiditis, Hashimoto's thyroiditis,
hyperthyroidism and hypothyroidism) , parathyroid disorders (including
hyperparathyroidism and hypoparathyroidism) s me and
, Gushing'
obesity;
- autoimmune es like systemic lupus erythemotosus;
- proliferative diseases including paraganglioma tumors, prostate cancer
and bone tumors;
- traumatic injuries including sports injuries, muscle injuries, tendon
wounds and bone fractures; and
- veterinary uses (e.g. immobilisation of mammals, equine colic, animal
achalasia or animal muscle ).The use of the pharmaceutical
composition according to the invention in cosmetics or esthetics is also an
aspect of the present invention, for example for treating or preventing treat or
prevent skin wrinkles, in particular facial es such as facial frown lines,
wrinkles of the contour of the eye, glabellar frown lines, downturned mouth.
The pharmaceutical composition according to the ion can also be used
in aesthetic medicine (that is for improving cosmetic appearance), in particular
for treating or ting skin wrinkles, in particular facial wrinkles such as
facial frown lines, wrinkles of the contour of the eye, glabellar frown lines,
downturned mouth, wrinkles of the neck (platysmal bands), wrinkles of the chin
lis, peau ge, dimpled chin), forehead lines, “scratched skin”
wrinkles, nasal lift treatment or sleep lines.
Unless defined otherwise, all technical and scientific terms used herein have
the same meaning as commonly understood by one of ry skill in the art
to which this disclosure belongs. ton, et al., DICTIONARY OF
MICROBIOLOGY AND LAR BIOLOGY, 20 ED., John Wiley and
Sons, New York (1994), and Hale & Marham, THE HARPER COLLINS
DICTIONARY OF BIOLOGY, Harper Perennial, NY (1991) provide one of skill
with a general dictionary of many of the terms used in this disclosure.
This disclosure is not limited by the exemplary methods and materials
disclosed herein, and any methods and materials similar or equivalent to those
described herein can be used in the ce or testing of embodiments of this
disclosure. Numeric ranges are ive of the numbers defining the range.
Unless othenNise indicated, any c acid sequences are written left to right
in 5' to 3' orientation; amino acid sequences are written left to right in amino to
carboxy orientation, respectively.
The headings provided herein are not limitations of the various aspects or
embodiments of this disclosure which can be had by reference to the
specification as a whole. Accordingly, the terms defined immediately below
are more fully defined by reference to the specification as a whole.
Amino acids are ed to herein using the name of the amino acid, the three
letter abbreviation or the single letter abbreviation.
2017/066361
As used , the term “amino acid sequence” is synonymous with the term
“polypeptide” and/or the term “protein”. In some instances, the term “amino
acid sequence” is synonymous with the term “peptide”. In some instances, the
term “amino acid sequence” is synonymous with the term “enzyme”.
The term in", as used herein, includes proteins, polypeptides, and
peptides.
The terms "protein" and "polypeptide" are used hangeably herein. In the
present sure and claims, the conventional one-letter and letter
codes for amino acid residues may be used. The 3-letter code for amino acids
as defined in conformity with the IUPACIUB Joint Commission on Biochemical
lature (JCBN). It is also understood that a polypeptide may be coded
for by more than one nucleotide sequence due to the degeneracy of the
genetic code.
Other definitions of terms may appear throughout the specification. Before the
exemplary embodiments are described in more detail, it is to understand that
this disclosure is not limited to particular embodiments described, as such may,
of , vary. It is also to be understood that the terminology used herein is
for the purpose of describing particular embodiments only, and is not intended
to be limiting, since the scope of the present disclosure will be limited only by
the ed claims.
Where a range of values is provided, it is understood that each intervening
value, to the tenth of the unit of the lower limit unless the context clearly
dictates otherwise, n the upper and lower limits of that range is also
specifically disclosed. Each smaller range between any stated value or
intervening value in a stated range and any other stated or intervening value
in that stated range is encompassed within this disclosure. The upper and
lower limits of these smaller ranges may independently be included or
excluded in the range, and each range where either, neither or both limits are
included in the smaller ranges is also encompassed within this disclosure,
subject to any specifically excluded limit in the stated range. Where the stated
range includes one or both of the limits, ranges excluding either or both of
those included limits are also included in this disclosure.
WO 02348 2017/066361
It must be noted that as used herein and in the appended claims, the singular
forms "a", "an", and "the" include plural referents unless the context clearly
dictates vise. Thus, for example, reference to "a clostridial neurotoxin"
es a plurality of such candidate agents and reference to "the clostridial
neurotoxin" includes reference to one or more clostridial neurotoxins and
equivalents thereof known to those skilled in the art, and so forth.
The publications discussed herein are provided solely for their disclosure prior
to the filing date of the present application. Nothing herein is to be construed
as an admission that such publications constitute prior art to the claims
appended hereto.
The invention will now be bed, by way of example only, with reference
to the following Figures and es.
BRIEF DESCRIPTION OF THE GS
Embodiments of the invention will now be described, by way of example only,
with reference to accompanying drawings, in which:
Figure 1 shows the relative amounts of full length activated botulinum
neurotoxin (endonegative) and truncated activated botulinum neurotoxin
heavy chains following incubation of unactivated num neurotoxin
samples at pH 8 and a protein concentration of 0.55 mg/mL at 20 °C with
recombinant porcine trypsin (Roche) at final concentrations of 0.3 and 0.4
ug/mL respectively. Samples were removed for analysis by SDS—PAGE. Each
SDS—PAGE sample was analysed by densitometry.
Figure 2 shows analysis by SDS—PAGE under reducing conditions, after
activation of single chain endonegative num neurotoxin E (0.55 mg/mL)
at pH 8.0 with bovine trypsin -Aldrich) at various concentrations and
tion for 8 hours at 20 °C with. (Lane 1: Molecular weight marker; lane 2:
-20 °C control; lane 3: +20 °C control; lane 4: 0.2 ug/mL trypsin; lane 5: 0.4
ug/mL trypsin; lane 6: 0.6 ug/mL trypsin; lane 7: 0.8 ug/mL trypsin; lane 8: 1.0
ug/mL trypsin).
2017/066361
Figure 3 shows the respective percentages of endonegative num
neurotoxin E heavy chain (HC), light chain (LC) and truncated heavy chain
(tHC) after tion with recombinant bovine trypsin at pH 6.5, 7.0, 7.5 and
7.8 and incubation for 16 hours at 20°C toxinconcentration: 0.55 mg/mL,
recombinant bovine trypsin (Sigma-Aldrich) concentration: 1,5 ug/mL).
Samples were analysed by SDS-PAGE under reducing conditions by
densitometry.
Figure 4 shows that the separation of full-length, di-chain num neurotoxin
E (endonegative) from truncated di-chain botulinum neurotoxin can be
achieved following activation with bovine trypsin and separation using a
ceramic hydroxyapatite type II tography column. The elution of full
length di-chain botulinum neurotoxin and truncated di-chain num
neurotoxin from the column was monitored by online A280nm readings, selected
fractions containing only full length di-chain botulinum neurotoxin were pooled
and analysed by SDS-PAGE under reducing and non-reducing conditions.
Figure 5: protein sequence of BoNT/A - UniProtKB Accession Number
P10845 (SEQ ID NO: 2).
Figure 6: protein sequence of BoNT/B - UniProtKB Accession Number
P10844 (SEQ ID NO: 3).
Figure 7: protein sequence of BoNT/C - UniProtKB Accession Number
P18640 (SEQ ID NO: 4).
Figure 8: n sequence of BoNT/D - UniProtKB Accession Number
P19321 (SEQ ID NO: 5).
Figure 9: protein sequence of BoNT/E - UniParc |.D UPI00000010A3 (SEQ ID
NO: 6).
Figure 10: protein sequence of BoNTiF - UniProtKB ion Number
YP_001390123 (SEQ ID NO: 7).
Figure 11: protein sequence of BoNT/G - UniProtKB Accession Number
Q60393 (SEQ ID NO: 8).
WO 02348
Figure 12: protein sequence of TeNT - UniProtKB Accession Number P04958
(SEQ ID NO: 9).
Figure 13: protein sequence of bovine trypsin (SEQ ID NO: 1).
EXAMPLES
All publications mentioned in the above specification are herein incorporated
by reference. Various modifications and variations of the described methods
and system of the present invention will be nt to those skilled in the art
without ing from the scope and spirit of the present invention. Although
the present invention has been described in connection with specific preferred
embodiments, it should be understood that the invention as claimed should not
be unduly limited to such specific ments. Indeed, various cations
of the described modes for ng out the invention which are obvious to
those skilled in biochemistry and biotechnology or related fields are intended
to be within the scope of the following claims.
Example 1
Single chain botulinum neurotoxin E (endonegative) samples at pH 8 and a
protein concentration of 0.55 mg/mL were incubated at 20 °C with recombinant
porcine trypsin (Roche) at final concentrations of 0.3 and 0.4 ug/mL. Samples
were removed for analysis by SDS-PAGE under reducing conditions every 30
s up to 6 hours and every 60 minutes afterwards up to 9 hours. Each
SDS-PAGE s was analysed by densitometry to determine the ve
amounts of full length di-chain botulinum neurotoxin, truncated di-chain
botulinum neurotoxin heavy chains and single chain num neurotoxin. The
values for the full length in botulinum neurotoxin and truncated di-chain
botulinum neurotoxin were then plotted on a chart (Figure 1). Truncated di-
chain botulinum neurotoxin occurs before full activation of the botulinum
neurotoxin is achieved when contacted with porcine trypsin.
Example 2 — Activation with different concentrations of bovine trypsin
Single chain botulinum neurotoxin E (endonegative) samples at pH 8.0 with a
n concentration of 0.55 mg/mL were ted at 20 °C with bovine
trypsin (Sigma-Aldrich) at final concentrations of 0.2, 0.4, 0.6, 0.8 and 1.0
ug/mL respectively. Samples were removed for analysis by SDS-PAGE under
reducing conditions after 8 hours. The results, ted in figure 2, show that
heavy chain truncation was observed in each sample before complete
activation had been achieved.
Example 3 — Activation with bovine trypsin at different pH
Single chain botulinum neurotoxin E (endonegative) samples at pH 6.5, 7.0,
7.5 and 7.8, with a protein concentration of 0.55 mg/mL were incubated at 20
°C with recombinant bovine trypsin (Sigma—Aldrich) at a final tration of
1,5 ug/mL. Samples were removed for analysis by SDS-PAGE under reducing
conditions after 16 hours. Each SDS-PAGE sample was analysed by
densitometry to determine the relative amounts of truncated di—chain botulinum
oxin heavy chain. The results are presented in figure 3 and table 1).
Truncated di-chain botulinum neurotoxin formation occurs more readily at
higher pH.
Table 1 — Percentage of ted heavy chain at different pH (<LOD: below
limit of detection)
pH Truncated Heavy Chain (%)
6.5 <LOD
7.0 <LOD
7.5 8.9
7.8 12.1
Example 4 — Purification of activated neurotoxin after activation with bovine
trypsin
26 mg of total protein ning endonegative BoNT/E that had been ted
by incubation with 78.57 pg Trypzean (bovine trypsin) for 18 hours at 20 °C
was applied to a 5 mL c hydroxyapatite type II column. The column was
washed with binding buffer (25 mM sodium phosphate, pH 6.5) and then eluted
over 35 column volumes increasing the sodium phosphate concentration with
a linear gradient using binding buffer and elution buffer (500 mM sodium
phosphate pH 6.5), collecting 2.5 mL fractions. The elution of full length, di-
chain botulinum neurotoxin and truncated di-chain botulinum neurotoxin from
the column was red by online A280nm readings, selected fractions
containing only full length di-chain num oxin were pooled and
analysed by GE under reducing and non-reducing conditions (Fig. 4).
Example 5 — Full length activated Botulinum neurotoxin E preparation
A botulinum neurotoxin E inoculum E.coli culture was prepared by thawing a
seed bank vial and inoculating a shake flask containing 100 mL modified
ic Broth (mTB). The flasks were then ted at 25 °C for 17 hours in
a shaking incubator. The inoculum culture was used to inoculate five shake
flasks, each containing 1 L of mTB. The cells were cultivated at 37 °C in a
shaking incubator into exponential growth phase; the temperature of the
cultures reduced to 16 °C; and the cultures were induced by adding isopropyl
B-Dthiogalactopyranoside (IPTG) to a final concentration of 0.1 mM. The
cells were harvested 20 hours post-induction by tangential flow filtration (TFF)
using a hollow fibre membrane. The culture was first concentrated five-fold
and then diafiltered with five s of lysis buffer (100 mM sodium
phosphate, 100 mM NaCl, 1.3 M (NH4)ZSO4, pH 7.8).
The resulting cell paste slurry was homogenised by two passes through a
mechanical cell disrupter. The insoluble cell debris was sedimented by
centrifugation and the supernatant was recovered and applied to a column
packed with Butyl Sepharose 4 FF (GE iences), which was washed with
g buffer (100 mM sodium phosphate, 100 mM NaCl, 1.25 M (NH4)2SO4,
pH 7.8). The unactivated botulinum neurotoxin E was eluted from the column
using three step gradients with the following mixtures of binding and elution
(100 mM sodium ate, 100 mM NaCl, pH 7.8) buffers, with the product
eluted in step 2.
Step 1 88 % loading ; 12 % elution buffer
Step 2 58 % loading buffer; 42 % elution buffer
Step 3 100 % elution buffer
The material from step 2 was then concentrated approximately two-fold by TFF
using a hollow fibre membrane and then diafiltered with 10 volumes of 25 mM
sodium phosphate pH 6.5 buffer. After diafiltration any insoluble material in
the retentate was sedimented by centrifugation and the supernatant applied to
a column packed with Q Sepharose HP (GE Lifesciences) in a negative
chromatography step. The flowthrough containing the unactivated botulinum
neurotoxin was collected and the column was washed with 25 mM sodium
phosphate pH 6.5 to maximize product recovery.
The flowthrough was then diluted to a total protein concentration of 0.5 mg/ml
with 25 mM sodium phosphate pH 6.5 and ted with 7.27 USP units/mL
recombinant bovine trypsin (TrypZean®) for 21 hours at room temperature.
After tion the activated botulinum neurotoxin E was then applied to a
ceramic yapatite type II column, which was washed with binding buffer
(25 mM sodium phosphate pH 6.5). The activated botulinum neurotoxin was
eluted from the column with a linear nt using binding buffer and elution
buffer (500 mM sodium phosphate pH 6.5).
Fractions containing full-length, activated num neurotoxin were pooled
and applied to a column packed with Benzamidine Sepharose FF (high-sub)
(GE Lifesciences) in a negative chromatography step. The flowthrough
containing the full-length, activated num neurotoxin was collected and the
column was washed with loading buffer (110 mM sodium phosphate, pH 6.5)
to maximize product recovery. The flowthrough was diafiltered by TFF using
a hollow fibre cartridge into the final storage buffer with 5 s of 25 mM
sodium phosphate, 100 mM NaCl, pH 6.5.
W0 2018;002348 2017/066361
Claims (17)
1. Method for ing an activated idial neurotoxin, comprising ting a single chain clostridial neurotoxin with an activation enzyme until at least 90% of the single chain clostridial neurotoxin is converted into a di- chain clostridial neurotoxin.
2. Method according to claim 1, wherein said activation enzyme is a trypsin.
3. Method according to claim 2, wherein said trypsin is a bovine trypsin and wherein said bovine trypsin has an amino acid sequence which has at least 90% identity to SEQ ID NO: 1. 10
4. Method according to claim 3, wherein said bovine trypsin is selected from a native trypsin obtained from bovine pancreas and a recombinant bovine trypsin.
5. Method according to claim 3 or 4, n said step of contacting said single chain clostridial neurotoxin with a bovine n is performed at a pH 15 of between 5 and 7,5, preferably between 6 and 7, for example at a pH of approximatively 6,5.
6. Method according to claim 5, wherein the step of contacting the single chain clostridial neurotoxin with said bovine trypsin is d out at room temperature at a pH between 6 and 7 for a duration of 15 to 25 hours, and 20 wherein the concentration of said bovine trypsin is between 0.5 and 3 pg per mg of clostridial neurotoxin.
7. Method according to any one of claims 1 to 6, further comprising a step of removing truncated clostridial neurotoxin. W0 2018;002348
8. Method according to claim 7, wherein said step of removing truncated clostridial neurotoxin ses contacting said activated clostridial neurotoxin with a mixed mode chromatography resin.
9. Method according to any one of claims 1 to 8, wherein said idial oxin is selected from BoNT/A, BoNT/B, BoNT/C, BoNT/D, BoNT/E, BoNT/F, BoNT/G and TeNT.
10. Method according to any one of claims 1 to 9, wherein said clostridial neurotoxin is a modified botulinum neurotoxin as compared to a wild-type botulinum neurotoxin, eg a mutated clostridial neurotoxin, a chimeric 10 clostridial neurotoxin or a retargeted clostridial neurotoxin.
11. Method according to any one of claims 1 to 10, wherein said idial neurotoxin ses a BoNT/E activation loop.
12. Method according to anyone of claims 1 to 11, wherein said clostridial neurotoxin is a . 15
13. Method according to any one of claims 1 to 12, wherein said single chain clostridial neurotoxin is obtained by expressing a gene encoding said single chain clostridial neurotoxin in a heterologous host cell, preferably in E. coli.
14. An active di-chain clostridial oxin obtainable by the method of any 20 one of claims 1 to 13.
15. A pharmaceutical composition comprising an active di-chain clostridial neurotoxin according to claim 14, wherein said composition is essentially free of single chain idial neurotoxin.
16. A pharmaceutical composition ing to claim 15 for use in therapy, 25 eg for treating ophtalmological disorders, movement disorders, otorhinolaryngological disorders, gastrointestinal disorders, urogenital W0 2018l002348 disorders, dermatological disorders, pain disorders, inflammatory disorders, secretory ers, respiratory disorders, hypertrophic disorders, articular ers, endocrine disorders, autoimmune diseases, proliferative diseases, traumatic injuries, veterinary uses. 5
17. Use of a pharmaceutical ition according to claim 15 in cosmetics or esthetics.
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP16177651.3 | 2016-07-01 |
Publications (1)
Publication Number | Publication Date |
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NZ749400A true NZ749400A (en) |
Family
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