U.S. patent application number 12/908647 was filed with the patent office on 2011-04-21 for methods and systems for purifying non-complexed botulinum neurotoxin.
This patent application is currently assigned to Revance Therapeutics, Inc.. Invention is credited to Curtis L. Ruegg.
Application Number | 20110092682 12/908647 |
Document ID | / |
Family ID | 43879799 |
Filed Date | 2011-04-21 |
United States Patent
Application |
20110092682 |
Kind Code |
A1 |
Ruegg; Curtis L. |
April 21, 2011 |
Methods and Systems for Purifying Non-Complexed Botulinum
Neurotoxin
Abstract
Methods and systems for chromatographically purifying a
botulinum neurotoxin are provided. These methods and systems allow
for efficient purification of a non-complexed form of the botulinum
neurotoxin in high purity and yield that can be used as an active
ingredient in pharmaceutical preparations.
Inventors: |
Ruegg; Curtis L.; (Redwood
City, CA) |
Assignee: |
Revance Therapeutics, Inc.
Newark
CA
|
Family ID: |
43879799 |
Appl. No.: |
12/908647 |
Filed: |
October 20, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61253810 |
Oct 21, 2009 |
|
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Current U.S.
Class: |
530/416 |
Current CPC
Class: |
C12Y 304/24069 20130101;
C12N 9/52 20130101 |
Class at
Publication: |
530/416 |
International
Class: |
C07K 1/18 20060101
C07K001/18 |
Claims
1. A method for purifying a non-complexed botulinum toxin, the
method comprising: (i) providing a crude non-complexed botulinum
toxin; (ii) loading the crude non-complexed botulinum toxin on an
anion exchange column so as to permit capture of the non-complexed
botulinum toxin by the anion exchange column; (iii) eluting the
non-complexed botulinum toxin from the anion exchange column to
give an eluent comprising the non-complexed botulinum toxin; (iv)
loading a cation exchange column with the eluent from the anion
exchange column so as to permit capture of the non-complexed
botulinum toxin by the cation exchange column; and (v) eluting
purified non-complexed botulinum toxin from the cation exchange
column.
2. The method according to claim 1, wherein the crude non-complexed
botulinum toxin is obtained by obtaining a sample comprising
botulinum toxin complex; loading a hydrophobic interaction column
with the sample so as to permit capture of the botulinum toxin
complex by the hydrophobic interaction column; eluting the
botulinum toxin complex from the hydrophobic interaction
chromatography column; and dissociating the botulinum toxin complex
to obtain a mixture comprising the crude non-complexed botulinum
toxin.
3. The method according to claim 2, wherein the sample is a
supernatant or filtrate comprising the botulinum toxin complex.
4. The method according to claim 2, wherein the sample is obtained
by: subjecting a fermentation culture comprising the botulinum
toxin to acid precipitation to obtain an acid precipitate; and
performing tangential flow filtration on the precipitate to
concentrate precipitate.
5. The method according to claim 2, wherein the sample is obtained
by subjecting an insoluble fraction of a fermentation culture to
tangential flow filtration.
6. The method according to claim 2, wherein the sample is subjected
to a nuclease digestion before loading on the hydrophobic
interaction column.
7. The method according to claim 6, wherein the nuclease is derived
from an animal product free process.
8. The method according to claim 1, wherein the method is
substantially animal product free.
9. The method according to claim 1, wherein the purified
non-complexed botulinum toxin comprises at least one of botulinum
toxin type A, B, C.sub.1, D, F, F and G.
10. The method according to claim 1, wherein the purified
non-complexed botulinum toxin comprises a botulinum toxin type
A.
11. The method according to claim 1, wherein the purified
non-complexed botulinum toxin is at least 95% pure.
12. The method according to claim 1, wherein the purified
non-complexed botulinum toxin has an activity of at least 200
LD.sub.50 units/ng.
13. The method according to claim 1, wherein the method produces a
yield of at least about 2 mg/L fermentation culture.
14. The method according to claim 1 wherein the anionic column is
selected from the group consisting of a Q Sepharose HP, Q Sepharose
Fast Flow, and Q XL Sepharose column, and wherein the cationic
column is selected from the group consisting of a SP Sepharose, SP
Sepharose HP, SP Sephrose Fast Flow, Mono S, Source-S, Source-30S,
and Source-15S column.
15. The method according to claim 1 wherein a buffer for loading
the non-complexed botulinum toxin onto the anionic column is
selected from the group consisting of Tris, bis-Tris,
triethanolamine, and N-methyl diethanolamine.
16. The method according to claim 15 wherein the buffer is used at
a pH from 7.4 to 8.2.
17. The method according to claim 1 wherein a buffer for loading
the non-complexed botulinum toxin onto the cationic column is
selected from the group consisting of sodium phosphate, MES, and
HEPES.
18. The method according to claim 17 wherein the buffer is used at
a pH from 6.0 to 7.0
19. The method according to claim 1 wherein pH of the anionic
column is from 7.4 to 8.2.
20. The method according to claim 1 wherein pH of the cationic
column is from 6.0 to 7.0.
21. The method according to claim 1 wherein a gradient for eluting
the non-complexed botulinum toxin from the anionic column is
selected from the group consisting of an ascending gradient of
sodium chloride and an ascending gradient of potassium
chloride.
22. The method according to claim 21 wherein the gradient is used
at a pH from 7.4 to 8.4.
23. The method according to claim 1 wherein a gradient for eluting
the non-complexed botulinum toxin from the cationic column is
selected from the group consisting of an ascending gradient of
sodium chloride and an ascending gradient of potassium
chloride.
24. The method according to claim 23 wherein the gradient is used
at a pH from 6.0 to 7.0.
Description
[0001] This application claims the benefit of priority to U.S.
Provisional Application No. 61/253,810, filed on Oct. 21, 2009. The
contents of this U.S. Provisional Application are hereby
incorporated by reference in their entirety.
FIELD OF THE INVENTION
[0002] This invention relates generally to chromatographic methods
and systems for purifying free botulinum neurotoxin from cell
cultures to produce a high purity, high potency product.
BACKGROUND OF THE INVENTION
[0003] Botulinum toxin is a neurotoxic protein produced by the
bacterium Clostridium botulinum, as well as other Clostridial
species, such as Clostridium butyricum, and Clostridium baraffi.
The toxin blocks neuromuscular transmission and causes a
neuro-paralytic illness in humans and animals, known as botulism.
C. botulinum and its spores commonly occur in soil and putrefying
animal carcasses, and can grow in improperly sterilized or
improperly sealed food containers, which are the cause of many
botulism cases. Botulism symptoms can include difficulty walking,
swallowing, and speaking, and can progress to paralysis of the
respiratory muscles and finally death.
[0004] Botulinum toxin type A is the most lethal natural substance
known to man. In addition to serotype A, six other generally
immunologically distinct botulinum toxins have been characterized,
namely botulinum toxin serotypes B, C.sub.1, D, E, F, and G. The
different serotypes can be distinguished by neutralization with
type-specific antibodies and vary in severity of paralysis they
evoke and the animal species they mostly affect. The molecular
weight of the botulinum toxin protein molecule, for each of the
known botulinum toxin serotypes, is about 150 kD, composed of an
about 100 kD heavy chain joined to an about 50 kD light chain.
Nonetheless, the botulinum toxins are released by Clostridial
bacteria as complexes of the 150 kD toxin with one or more
non-toxin proteins. For example, botulinum toxin type A exists as
900 kD, 500 kD and 300 kD complexes (approximate molecular
weights).
[0005] Despite the known toxic effects, Botulinum toxin type A is
clinically used to treat a variety of indications, including, e.g.,
neuromuscular disorders characterized by skeletal muscle
hyperactivity. For example, BOTOX.RTM. is the trademark of a
botulinum toxin type A complex available commercially from
Allergan, Inc., of Irvine, Calif. Botulinum toxin type A finds use,
for example, in the treatment of essential blepharospasm,
strabismus, cervical dystonia, and glabellar line (facial) wrinkles
Other serotypes also have been used clinically. A botulinum toxin
type B, for example, has been indicated for use in treating
cervical dystonia. The botulinum toxins are believed to bind with
high affinity to the presynaptic membrane of motor neurons,
translocate into the neuron, and thereafter block the presynaptic
release of acetylcholine.
[0006] The botulinum toxin for clinical use is typically isolated
from cell culture and various purification approaches have been
used. Historically, the toxin is purified in complexed form by a
series of precipitation and tangential flow filtration steps. See,
e.g., Schantz E. J., et al., Properties and use of botulinum toxin
and other microbial neurotoxins in medicine, Microbiol Rev 1992
March 56(1):80-99. Such approaches have provided relatively low
yields, however, typically less than about 10%. Other approaches
have used size exclusion, ion exchange, and/or affinity
chromatography. See, e.g., Schmidt J. J., et al., Purification of
type E botulinum neurotoxin by high-performance ion exchange
chromatography, Anal. Biochem. 1986 July; 156(1):213-219; Simpson
L. L., et al., Isolation and characterization of the botulinum
neurotoxins, Harsman S, ed. Methods in Enzymology. Vol. 165,
Microbial Toxins: Tools in Enzymology San Diego, Calif.: Academic
Press; vol 165: pages 76-85 (1988); Kannan K., et al., Methods
development for the biochemical assessment of Neurobloc (botulinum
toxin type B), Mov Disord 2000; 15(Suppl 2):20 (2000); Wang Y. C.,
The preparation and quality of botulinum toxin type A for injection
(BTXA) and its clinical use, Dermatol Las Faci Cosm Surg 2002; 58
(2002); and U.S. Pat. Appl. Publ. No. 2003/0008367.
[0007] Still other approaches have focused on just one of the
toxin's heavy or light chains, rather than a complete and
biologically active botulinum toxin protein. For example, one of
the chains is individually synthesized by recombinant means. See,
e.g., Zhou L., et al., Expression and purification of the light
chain of botulinum neurotoxin A: A single mutation abolishes its
cleavage of SNAP-25 and neurotoxicity after reconstitution with the
heavy chain, Biochemistry 1995; 34(46):15175-81 (1995); and Johnson
S. K., et al., Scale-up of the fermentation and purification of the
recombination heavy chain fragment C of botulinum neurotoxin
serotype F, expressed in Pichia pastoris, Protein Expr and Purif
2003; 32:1-9 (2003). These approaches, however, require extra steps
to reform a complete and biologically active botulinum toxin
protein.
[0008] A more recent approach involves the use of hydrophobic
interaction chromatography, mixed mode, and/or ion exchange
chromatography to purify a botulinum toxin as a complex. See, e.g.,
U.S. Pat. Nos. 7,452,697 and 7,354,740, which are hereby
incorporated by reference.
[0009] Accordingly, there is a need in the art for improved
purification methods for isolating complete botulinum toxin
proteins in stable, biologically active, but non-complexed forms.
It is therefore an object of the invention to provide compositions
and methods addressing these and other needs.
[0010] The foregoing discussion is presented solely to provide a
better understanding of the nature of the problems confronting the
art and should not be construed in any way as an admission as to
prior art nor should the citation of any reference herein be
construed as an admission that such reference constitutes "prior
art" to the instant application.
SUMMARY OF THE INVENTION
[0011] This invention relates to systems and methods for purifying
a non-complexed botulinum toxin. In one embodiment, the method
comprises purifying crude non-complexed botulinum toxin to obtain a
purified non-complexed botulinum toxin. In this embodiment, the
method comprises loading an anion exchange column with the crude
non-complexed botulinum toxin to capture the non-complexed
botulinum toxin on the anion exchange column; eluting the
non-complexed botulinum toxin with buffer to give an eluent
comprising the non-complexed botulinum toxin; loading a cation
exchange column with the eluent from the anion exchange to column
to permit capture of the non-complexed botulinum toxin; and eluting
the non-complexed botulinum toxin with another buffer to give an
eluent, thereby obtaining a purified non-complexed botulinum
toxin.
[0012] In certain embodiments, the botulinum toxin complex is
itself obtained by a number of chromatography steps. In some
embodiments, a method for obtaining the botulinum toxin complex
comprises obtaining a sample comprising a botulinum toxin complex;
loading a hydrophobic interaction column with the sample to permit
capture of the toxin, wherein the captured botulinum toxin
comprises a complexed botulinum toxin; and eluting the complexed
botulinum toxin. The non-complexed botulinum toxin is then
dissociated from the complex and the non-complexed botulinum toxin
is purified according to the method described above. In some
embodiments, the sample is obtained by subjecting a fermentation
culture comprising botulinum toxin to acid to obtain an acid
precipitate, which may be subjected to additional
pre-chromatography purification steps, non-limiting examples of
which include tangential flow filtration to concentrate the
insoluble material of the precipitate, nuclease digest, clarifying
centrifugation and/or filtration.
[0013] In some embodiments, the sample is subjected to a nuclease
digestion before loading on the hydrophobic interaction column.
Preferably, the nuclease is derived in an animal-product-free
process, and even more preferably the entire purification process
is animal product free or at least substantially animal product
free.
[0014] In some embodiments, the sample to be used in the
chromatographic separations is preferably a supernatant or filtrate
fraction.
[0015] The purified non-complexed botulinum toxin comprises at
least one of botulinum toxin type A, B, C.sub.1, D, F, F and G, and
preferably a botulinum toxin type A having a molecular weight of
about 150 kD. In some preferred embodiments, the purified
non-complexed botulinum toxin is at least 98% pure; and/or has an
activity of at least 200 LD.sub.50 units/ng. In some embodiments,
the method produces a yield of at least about 2 mg/L fermentation
culture. In other embodiments, the method produces a yield of about
1 to about 2 mg/L fermentation culture.
[0016] These and other aspects of the invention will be better
understood by reference to the following detailed description of
the invention.
BRIEF DESCRIPTION OF THE FIGURES
[0017] FIG. 1 is a summary flow chart comparing one embodiment of a
process according to the instant invention for directly purifying a
non-complexed botulinum toxin (FIG. 1A) with a process for
purifying a complexed botulinum toxin (FIG. 1B).
DETAILED DESCRIPTION
[0018] This invention relates to systems and methods for purifying
a non-complexed botulinum toxin. In certain embodiments, the method
comprises purifying a crude non-complexed botulinum toxin by
loading an anion exchange column with the crude non-complexed
botulinum toxin to permit capture of the non-complexed botulinum
toxin by the anion exchange column. Non-complexed botulinum toxin
is then eluted with buffer to give an eluent comprising the
non-complexed botulinum toxin. The eluent from the anion column is
loaded on a cation exchange column to permit capture of the
non-complexed botulinum toxin and the purified non-complexed
botulinum toxin is eluted with buffer, thereby obtaining a purified
non-complexed botulinum toxin.
[0019] In some embodiments, the invention provides for the
purification of non-complexed botulinum toxin in a relatively small
number of steps to produce a high yield, high purity, and high
potency product. Processes and systems within the scope of the
invention can be used to efficiently produce a stable but
non-complexed botulinum toxin from fermentation cultures. In other
embodiments, the method further comprises providing a sample
comprising botulinum toxin complex and loading a hydrophobic
interaction column with the sample so as to permit capture of the
botulinum toxin complex by the hydrophobic interaction column. The
botulinum toxin complex is then eluted from the column with buffer.
Crude non-complexed botulinum toxin is dissociated from the
botulinum toxin complex to obtain a mixture comprising the crude
non-complexed botulinum toxin. In this embodiment, the mixture
comprising crude non-complexed botulinum toxin is purified to
obtain pure or substantially pure botulinum toxin according to the
method described above.
[0020] One aspect of this invention is the recognition that a
pharmaceutical composition comprising non-complexed botulinum toxin
as an active ingredient can provide greater purity compared to one
comprising a complexed form. Non-toxin proteins typically
associated with a botulinum toxin complex can account for about 90%
by weight of the complex. Thus, providing a botulinum toxin as a
complex necessarily includes at least about 90% by weight of
impurities. In other words, at least about 80 to about 90% by
weight of the pharmaceutical composition will include cell-derived
impurities that are not part of the active molecule nor necessary
for its biological activity. Such impurities, however, represent
cell-derived materials that when administered to a patient may
increase the risk of unwanted immunological reactions to the drug;
may increase the risk of unwanted side effects; and/or may increase
the risk of transmission of pathogenic agents. In contrast, the
high purity of a non-complexed product, obtainable by methods and
systems described herein, reduces the amount of host cell
impurities that may remain in the pharmaceutical composition,
thereby reducing the attendant risks of unwanted reactions and/or
transmission. Accordingly, processes and systems described herein
can provide a botulinum toxin in a form more readily suited to the
preparation of safer, purer pharmaceutical compositions.
[0021] Moreover, unlike complexed forms, free botulinum toxin
prepared in accordance with the method described herein does not
need to be stabilized for storage in blood-derived products.
Botulinum toxin type A complex, for example, is typically
stabilized in an excipient comprising albumin, which is derived
from human blood. For example, BOTOX.RTM. consists of a purified
botulinum toxin type A complex, human serum albumin, and sodium
chloride packaged in vacuum-dried form. The same is true for
Dysport and Xeomin. While screenings reduce likelihood of
contamination with pathogenic agents, use of human blood in
pharmaceutical preparations generally increases the risk of
unwanted transmission of certain pathogenic agents, e.g., agents
which are not or cannot yet be screened out. In contrast, free
botulinum toxin prepared according to the instant invention can be
stably stored, as taught herein, in ammonium sulfate. Further, in
some preferred embodiments, methods and systems of the instant
invention are substantially, essentially, or entirely animal
product free, as discussed herein. The ability to also stably store
the toxin product substantially, essentially, or entirely
animal-product free, further reduces potential risks associated
with animal-derived products. Accordingly, processes and systems
described herein provide a botulinum toxin in a form particularly
suited to pharmaceutical applications in terms of safety, e.g.,
where the pharmaceutical composition may be prepared and stored
substantially, essentially, or entirely animal-product free.
[0022] In certain preferred embodiments, the processes and systems
described herein are scalable and/or cGMP compliant. Accordingly,
methods and systems described herein may be used on a commercial,
industrial scale, to produce non-complexed botulinum toxin for use,
e.g., in pharmaceutical compositions. A cGMP compliant process or
system refers to one that can comply with the regulatory
requirements for current good manufacturing practices, as required
by the U.S. Code of Federal Regulations. In some preferred
embodiments, the non-complexed botulinum toxin product is
particularly suited to large scale production due to its ease of
storage and usability, high activity, high purity, stability,
and/or improved safety.
[0023] "Botulinum toxin" as used herein refers to a neurotoxin
protein molecule that can be produced by a Clostridial bacterium,
as well as recombinantly produced forms thereof. A recombinant
botulinum toxin can have the light chain and/or heavy chain of the
toxin protein synthesized via recombinant techniques, e.g., by a
recombinant Clostridial and/or non-Clostridial species. "Botulinum
toxin" is used interchangeably herein with the related expressions
"botulinum neurotoxin," "neurotoxin" or simply "toxin." "Botulinum
toxin" encompasses any of the botulinum toxin serotypes A, B,
C.sub.1, D, E, F and G, and also encompasses both complexed and
non-complexed forms.
[0024] By "complexed form" is meant a botulinum toxin complex
comprising a botulinum toxin protein (i.e., the toxin molecule with
a molecular weight of about 150 kD) as well as at least one
associated native non-toxin protein. Non-toxin proteins that make
up the complexes typically include non-toxin hemagglutinin protein
and non-toxin non-hemagglutinin protein. Thus complexed forms may
comprise a botulinum toxin molecule (the neurotoxic component) and
one or more non-toxin hemagglutinin proteins and/or one or more
non-toxin non-hemagglutinin proteins. In certain embodiments, the
molecular weight of the complex is greater than about 150 kD. For
example, complexed forms of the botulinum toxin type A can have
molecular weights of about 900 kD, about 500 kD or about 300 kD.
Complexed forms of botulinum toxin types B and C.sub.1 can have a
molecular weight of 500 kD. Complexed forms of botulinum toxin type
D can have a molecular weight of about 300 kD or about 500 kD.
Finally, complexed forms of botulinum toxin types E and F can have
a molecular weight of about 300 kD.
[0025] "Non-complexed" botulinum toxin refers to an isolated, or
essentially or substantially isolated, botulinum toxin protein
having a molecular weight of about 150 kD. That is, "non-complexed"
forms exclude non-toxin proteins, such as non-toxin hemagglutinin
and non-toxin non-hemagglutinin proteins, normally associated with
the complexed form. "Non-complexed" botulinum toxin is used
interchangeably herein with "free" botulinum toxin. All the
botulinum toxin serotypes made by native Clostridium botulinum
bacteria are synthesized by the bacteria as inactive single chain
proteins which are then cleaved or nicked by proteases to become
neuroactive. The protein comprises an about 100 kD heavy chain
joined by a disulfide bond to an about 50 kD light chain.
[0026] Botulinum toxin complexes can be dissociated into toxin and
non-toxin proteins by various means, including, for example,
raising the pH to about 7.0, treating the complex with red blood
cells at a pH of about 7.3, and/or subjecting the complex to a
separation process, such as column chromatography in a suitable
buffer at a pH of about 7 to about 8.
[0027] The instant invention encompasses systems and methods that
enable purification of a non-complexed botulinum toxin, without
associated non-toxin proteins conventionally believed necessary
during the purification process to maintain stability. In preferred
embodiments, the methods and systems described herein facilitate
purification of the free botulinum toxin without loss of stability.
By "stability" or "stable" is meant that the botulinum toxin
protein molecule retains both the about 100 kD heavy chain and the
about 50 kD light chain, joined to each other by a disulfide bond,
and in a conformation that allows for biological activity.
[0028] In some embodiments, a particular system within the scope of
the present invention is operated in conjunction with a particular
method within the scope of the present invention. A system within
the scope of the present invention can comprise a plurality
(preferably consecutive series) of chromatography columns for use
with a corresponding plurality (preferably consecutive series) of
chromatography steps. Further, a system within the scope of the
instant invention may comprise a plurality (preferably a
consecutive series) of non-chromatography devices, such as
filtration and/or centrifugation apparatus, for use with a
corresponding plurality (preferably consecutive series) of
non-chromatography steps, e.g., as pre-chromatography steps.
[0029] In preferred embodiments, a process within the scope of the
present invention comprises obtaining a sample comprising botulinum
toxin from a fermentation culture; subjecting it to a number of
pre-chromatography purifications; and then passing it through a
plurality of chromatography columns to obtain a highly purified,
highly potent non-complexed botulinum toxin. Such a purified free
botulinum toxin finds use in the preparation of pharmaceutical
compositions comprising the free botulinum toxin as an active
ingredient.
[0030] The overall steps for both pre-chromatography and
chromatography processes for some preferred embodiments of the
instant invention are illustrated in FIG. 1A. For comparison, FIG.
1B shows a conventional method for obtaining purified botulinum
toxin complex. Briefly, FIG. 1B depicts a process involving depth
filtration of a fermentation culture, followed by tangential flow
filtration of the filtrate obtained (using 300 kD
ultramicrofiltration); followed by a clarifying centrifugation
step. The pellet (insoluble fraction) resulting from the
centrifugation step is then re-suspended in sodium chloride, and
loaded onto a hydrophobic interaction or ion exchange column. The
chromatographic purification step is repeated at least three times
to give a final eluent containing the 900 kD botulinum toxin type A
complex.
[0031] Fermentation and Acid Precipitation
[0032] As FIG. 1A illustrates, the non-complexed botulinum toxin is
generally purified from a fermentation culture. A "fermentation
culture" as used herein refers to a culture or medium comprising
cells, and/or components thereof, that are synthesizing and/or have
synthesized at least one botulinum toxin. For example, Clostridial
bacteria, such as Clostridium botulinum, may be cultured on agar
plates in an environment conducive to bacterial growth, such as in
a warm anaerobic atmosphere. The culture step typically allows
Clostridial colonies with desirable morphology and other
characteristics to be obtained. Selected cultured Clostridial
colonies then can be fermented in a suitable medium as a
fermentation culture. The cultured cells may include
non-Clostridial species as the host cells, such as E. coli or yeast
cells, that are rendered capable of biosynthesizing a botulinum
toxin by recombinant technology. Suitable fermentation culture
conditions can depend on the host cells used and are generally
known in the art.
[0033] In preferred embodiments, fermentation may be allowed to
progress to completion, such that cells are mature and have
biosynthesized a botulinum toxin. Growth of Clostridium botulinum
cultures is usually complete after about 24 to about 36 hours.
After a certain additional period of time, the bacteria typically
lyse and release into the medium the synthesized botulinum toxin
complex in a complexed form. For example, during a fermentation of
about 60 to about 96 hours, most Clostridium botulinum cells
undergo lysis and release botulinum toxin type A complex.
[0034] In some embodiments, the fermentation culture can comprise
one or more animal products, such as animal proteins, used in
conventional fermentation culture procedures. For example,
botulinum toxin can be produced by anaerobic fermentation of
Clostridium botulinum using a modified version of the well known
Schantz process (see e.g. Schantz E. J., et al., Properties and use
of botulinum toxin and other microbial neurotoxins in medicine,
Microbiol Rev 1992 March; 56(1):80-99; Schantz E. J., et al.,
Preparation and characterization of botulinum toxin type A for
human treatment, chapter 3 in Jankovic J, ed. Neurological Disease
and Therapy. Therapy with botulinum toxin (1994), New York, Marcel
Dekker; 1994, pages 41-49, and; Schantz E. J., et al., Use of
crystalline type A botulinum toxin in medical research, in: Lewis G
E Jr, ed. Biomedical Aspects of Botulism (1981) New York, Academic
Press, pages 143-50, each incorporated herein by reference). Both
the Schantz and the modified Schantz process for obtaining a
botulinum toxin make use of animal products, including
animal-derived-Bacto-Cooked Meat medium in the culture vial, and
casein in the fermentation media. Additionally, the Schantz toxin
purification makes use of DNase and RNase from bovine sources to
hydrolyze nucleic acids present in the fermentation culture.
[0035] However, administration of a pharmaceutical containing an
active ingredient that was purified using a process involving
animal-derived products can subject a patient to a potential risk
of receiving various pathogenic agents. For example, prions may be
present in a pharmaceutical composition comprising contaminating
animal-derived products, such as the prion responsible for
Creutzfeldt-Jacob disease. As another example, there is a risk of
transmitting a spongiform encephalopathy (TSE), such as a bovine
spongiform encephalopathy (BSE) when animal products are used in
the process of making a pharmaceutical composition. The use of a
botulinum toxin obtained via processes free of animal products,
however, reduces such risks. Therefore, in some preferred
embodiments, the invention provides a process that is free of
animal products, or essentially or substantially
animal-product-free (APF). "Animal product free", "essentially
animal product free", or "substantially animal product free"
encompasses, respectively, "animal protein free", "essentially
animal protein free", or "substantially animal protein free" and
respectively means the absence, essential absence, or substantial
absence, of products derived from animals, non-limiting examples of
which include products derived from blood or pooled blood. "Animal"
is used herein to refer to a mammal (such as a human), bird,
reptile, amphibian, fish, insect, spider or other animal species,
but excludes microorganisms, such as bacteria and yeasts.
[0036] An animal-product-free process (or a substantially or
essentially animal product-free-process) refers to a process that
is entirely, substantially, or essentially free of animal-derived
products, reagents and proteins, such as immunoglobulins, other
blood products, by-products, or digests; meat products, meat
by-products, meat digests; and milk or dairy products, by-products
or digests. Accordingly, an example of an animal-product free
fermentation culture procedure is a fermentation process, such as
bacterial culturing, which excludes blood, meat, and dairy
products, by-products, and digests. An animal-product-free
fermentation process for obtaining a non-complexed botulinum toxin
reduces the possibility of contamination with viruses, prions or
other undesirable agents, which can then accompany the toxin when
administered to humans.
[0037] Animal-product-free fermentation procedures using
Clostridium cultures are described, e.g., in U.S. Pat. Nos.
7,452,697 and 7,354,740, hereby incorporated by reference. For
example, the growth media for production of the botulinum toxin may
comprise vegetable-based products, instead of animal-derived
products, such as soy-based products and/or the debittered seed of
Lupinus campestris. Soy-based fermentation media for use in an
animal product free fermentation culture, for example, can comprise
a soy-based product, a source of carbon such as glucose, salts such
as NaCl and KCl, phosphate-containing ingredients such as
Na.sub.2HPO.sub.4 and KH.sub.2PO.sub.4, divalent cations such as
iron and magnesium, iron powder, amino acids such as L-cysteine and
L-tyrosine, and the like. Preferably, the soy is hydrolyzed soy and
the hydrolyzation has been conducted using enzymes not derived from
animals. Sources of hydrolyzed soy include but are not limited to
Hy-Soy (Quest International), Soy peptone (Gibco) Bac-soytone
(Difco), AMISOY (Quest), NZ soy (Quest), NZ soy BL4, NZ soy BL7,
SE50M (DMV International Nutritionals, Fraser, N.Y.), and SE50MK
(DMV).
[0038] As FIG. 1A illustrates, in certain embodiments a sample
comprising botulinum toxin is obtained from a fermentation culture.
For example, after a certain period of fermentation, in either
animal product free or non-animal product free media, botulinum
toxin complex is released into the medium and can be harvested by
precipitation. For example, in some embodiments, as in the
well-known Schantz process, the fermentation medium comprising the
botulinum toxin may be subjected to acid precipitation to encourage
the botulinum toxin complexes to associate with cell debris and
form an acid precipitate. In some particularly preferred
embodiments, about 3 M sulfuric acid solution may be added to the
fermentation culture to form the acid precipitate. Preferably, the
pH is reduced to about 3 to about 4, more preferably to about 3.2
to about 3.8, and even more preferably about 3.5. In some
embodiments, the culture temperature is also reduced, e.g., to
about below 25.degree. C., 24.degree. C., 23.degree. C., 22.degree.
C., 21.degree. C., or 20.degree. C. These conditions further
enhance the association of botulinum toxin complexes with cell
debris. The acid precipitate formed will comprise bound botulinum
toxin complexes and can be used as the starting material in further
purification steps, such as clarification steps; whereas the
filtrate is discarded.
[0039] In contrast, the conventional process depicted in FIG. 1B
does not include an acid precipitation step. That is, while the
purification procedure also begins with a fermentation culture
comprising a botulinum toxin complex, the culture medium is
subjected to depth filtration, and the filtrate, rather than the
cell debris, is used in subsequent purification steps. In the FIG.
1B process, the cell debris is discarded, rather than the filtrate,
whereas, as illustrated in FIG. 1A, the filtrate is discarded and
the cell debris (acid precipitate) is used for further purification
steps, e.g., in the pre-chromatography purifications discussed
below.
[0040] Pre-Chromatography Purifications
[0041] In some embodiments, the sample obtained from the
fermentation medium is subjected to one or more pre-chromatography
purifications. Pre-chromatography purifications can include at
least one of tangential flow filtration, nuclease digest, and
clarifying centrifugation and/or filtration. A non-limiting example
of a process flow containing pre-chromatography purification
contemplated by the invention is provided in FIG. 1A. As noted
above, in preferred embodiments, the pre-chromatography procedures
are carried out on a precipitate (or insoluble fraction) of a
fermentation culture comprising the botulinum toxin, rather than on
the fermentation culture itself or on a filtrate derived therefrom,
as in the process illustrated in FIG. 1B. That is, in preferred
embodiments of the invention, pre-chromatography (clarification)
steps start with the acid precipitate (insoluble fraction).
[0042] In some embodiments, the sample (acid precipitate or
insoluble fraction) comprising a botulinum toxin is subjected to
tangential flow filtration. Tangential flow filtration is a process
typically used to clarify, concentrate, and/or purify proteins. In
contrast to normal flow filtration, where fluid moves directly
towards a filter membrane under applied pressure, in tangential
flow filtration, the fluid moves tangentially along, or parallel
to, the surface of the membrane. Applied pressure serves to force a
portion of the fluid through the filter membrane, to the filtrate
side, while particulates and macromolecules too large to pass
through membrane pores are retained. Unlike normal flow filtration,
however, the retained components do not build up at the membrane
surface but are swept along by the tangentially flowing fluid. In
certain preferred embodiments, tangential flow filtration is used
to concentrate the insoluble material (cell debris) with which the
botulinum complex is associated, while permitting filtrate to pass
through the membrane pores. (See, e.g., FIG. 1A.) Tangential flow
filtration parameters, such as pore size, feed flow, applied
pressure, and the like, may be selected by those of skill in the
art to concentrate cell debris and to produce a more concentrated
sample comprising the botulinum toxin complex. In some particularly
preferred embodiments, for example, tangential flow filtration with
filters having a pore size of about 0.1 .mu.m may be used.
[0043] In some embodiments, the sample comprising a botulinum toxin
is subjected to nuclease digestion. Nuclease digestion can
facilitate removal of nucleic acid components with which the
Botulinum toxin complexes tend to associate. In certain preferred
embodiments, nuclease digestion follows tangential flow filtration
and is carried out on the concentrated cell debris obtained
therefrom. (See, e.g., FIG. 1A.) For example, the concentrated cell
debris sample may have its pH adjusted to allow nuclease activity
and may be incubated with one or more suitable nucleases, such as
DNases and/or RNases that digest (hydrolyze) DNA and/or RNA,
respectively. Depending on the nuclease enzyme used, suitable pH
may be about 5 to about 7, preferably about 6. In some embodiments,
benzamidine is used as a protease inhibitor to prevent proteolysis
of the toxin during nuclease digestion step. The nuclease used may
be derived from any suitable source, including animal sources
and/or non-animal sources.
[0044] In more preferred embodiments, the nuclease is obtained from
a non-animal source, to provide an animal-product-free nuclease and
an animal-product-free process. Accordingly, the instant invention
encompasses animal-product-free processes and systems (or
substantially or essentially animal product free processes and
systems) for purifying botulinum toxin which comprise use of a
nuclease. An animal-product-free nuclease may be made
recombinantly, e.g., using recombinant bacteria, yeasts, or other
suitable microorganisms, which have been transformed to express a
DNase and/or RNase for use in a nuclease digestion step according
to processes described herein. Nuclease digestion typically reduces
the nucleic acid content of the sample, as the host cell nucleic
acids are degraded and their removal is facilitated. For example,
hydrolyzed nucleic acids and other low molecular weight impurities
can be removed by further purification steps.
[0045] In certain embodiments, the sample comprising a botulinum
toxin may be subjected to clarifying centrifugation and/or
filtration. Clarifying centrifugation or filtration refers to
centrifugation or filtration steps used to remove gross elements,
such as whole and lysed cells and cell debris, from the sample,
resulting in a measurably clearer sample. In certain embodiments,
the centrifugation is performed at about 10,000.times.g to about
30,000.times.g, more preferably at about 15,000.times.g to about
20,000.times.g, and most preferably at about 17,700.times.g.
Clarifying filtration will typically comprise normal flow
filtration, also called "dead end" filtration, where fluid is moved
directly toward a filter media under applied pressure, and
particulates too large to pass through the filter pores accumulate
at the surface or within the media itself, while smaller molecules
pass through as the filtrate. In some particularly preferred
embodiments, the sample is mixed with ammonium sulfate and normal
flow filtration is performed using a filter with a pore size of
about 0.1 to about 0.3 .mu.m, and more preferably a pore size of
about 0.2 .mu.m. (See, e.g., FIG. 1A.) In certain particularly
preferred embodiments, one or more clarifying step(s) follow the
nuclease digestion step. In certain still more preferred
embodiments, one or more clarifying step(s) immediately precede
purification by chromatography.
[0046] Notably, in preferred embodiments, the clarified supernatant
or filtrate provides the botulinum toxin-containing sample for use
in further purification steps, such as the chromatography
purification steps, rather than the insoluble fraction, which is
discarded. This is in contrast with the process outlined in FIG.
1B, where the botulinum toxin complex is contained in the insoluble
fraction from pre-chromatography steps that do not involve acid
precipitation, such as e.g., as a centrifugation pellet, obtained
from pre-chromatography centrifugation, and the supernatant is
discarded.
[0047] Moreover, and again in contrast with the process outlined in
FIG. 1B, the pre-chromatography steps in some embodiments of the
invention do not require a tangential flow filtration step of a
filtrate obtained from fermentation culture. That is, the sample
used for chromatography purification in some embodiments of the
invention is not obtained by subjecting a soluble fraction of the
fermentation culture to tangential flow filtration. Rather, in
certain embodiments, the present invention uses insoluble material
(such as an acid precipitate), eliminating any step where a
fermentation culture filtrate is subjected to tangential flow
filtration in an attempt to concentrate soluble botulinum toxin
complexes. Thus, in preferred embodiments, the pre-chromatography
steps of the invention eliminate the need for any such step, by
instead using acid to precipitate the desired toxin complexes with
other insoluble material (cell debris).
[0048] Chromatography Purification Steps
[0049] FIG. 1A also illustrates chromatographic purification steps
according to certain embodiments of the instant invention.
According to one embodiment of the invention, chromatographic
methods for purifying a non-complexed botulinum toxin comprise
passing a sample comprising botulinum toxin through a plurality of
chromatography columns to obtain a highly purified, highly potent,
non-complexed form of the neurotoxin.
[0050] In certain embodiments, a complexed botulinum toxin is
separated from other cellular components using a hydrophobic
interaction column (see e.g., FIG. 1A). This column captures the
botulinum toxin in complexed form, while allowing impurities to
flow through the column. The column used may be any hydrophobic
interaction column known in the art suitable for such purpose, such
as Butyl Sepharose Fast Flow column or Phenyl Sepharose HP,
commercially available from GE Healthcare Life Sciences. In some
embodiments, the method further comprises conditioning the sample
for hydrophobic interaction chromatography before loading onto the
column. For example, for use in the Phenyl Sepharose HP column, the
sample may be combined with a 0.5M ammonium sulfate solution at pH
6, and 50 mM phosphate before loading. Other columns, buffers and
pH conditions that may be used include columns such as Phenyl
Sepharose Fast Flow high substitution, Phenyl Sepharose Fast Flow
low substitution, Butyl Sepharose, and Octyl Sepharose; buffers
such as acetate, citrate, MES, histidine, piperazine, and malonate,
each in the pH range of about 4.0 to about 7.0, more preferably
about 4.5 to about 6.5, and even more preferably about 5.5. Other
buffer and pH conditions may be determined to optimize yield from a
particular column used, as known in the art, based on the teachings
provided herein. Without wishing to be bound to theory, it is
believed that separation involves binding of the toxin complex to
resin at a pH below 7, to avoid dissociation at this step, while
allowing many cell-derived impurities to flow through, such as,
e.g., smaller proteins, nucleic acids, and the like.
[0051] For eluting the captured (bound) toxin from the hydrophobic
interaction column, a suitable buffer can be used, as known in the
art. In some particularly preferred embodiments, a descending
gradient of ammonium sulfate is used. The concentration range of
the descending gradient may be from about 0.6 M to about 0.0 M,
about 0.5 M to about 0.0 M, or about 0.4 M to about 0.0 M. Other
eluting buffers that may be used include, for example descending
gradients of sodium sulfate (Na.sub.2SO.sub.4); sodium chloride
(NaCl); potassium chloride (KCl); ammonium acetate (NH.sub.4OAc);
and the like. Fraction(s) containing a product peak can be
identified, as known in the art. The peak fraction is typically
found, e.g., when using ammonium sulfate, in a concentration range
of about 0.4 M to about 0.0 M; more preferably about 0.3 M to about
0.0 M; and most preferably about 0.25 M to about 0.0 M ammonium
sulfate, while the pH is kept at about 6 to maintain the complex.
That is, the fraction(s) containing the eluted botulinum toxin
complex can be identified and used in subsequent purification
steps.
[0052] In preferred embodiments, the botulinum toxin complex
obtained is caused to dissociate to give a non-complexed form. In
certain preferred embodiments, the dissociation step is performed
after the hydrophobic interaction chromatography step and/or before
subsequent chromatography steps (e.g., see FIG. 1A). Accordingly,
in some preferred embodiments, the instant invention encompasses
methods and systems where the chromatographic target molecule
differs from one chromatographic step to another. That is, in an
initial chromatographic step, the target comprises a botulinum
toxin complex, whereas in subsequent chromatographic steps, the
target comprises the free botulinum toxin, dissociated from
non-toxin proteins such as hemagglutinin and non-hemagglutinin
proteins. In contrast, the process outlined in FIG. 1B involves
chromatography steps that are all designed to purify botulinum
toxin complexes.
[0053] Dissociation of the botulinum toxin complex to produce the
non-complexed botulinum toxin protein may be achieved in a number
of ways, e.g., as known in the art and/or described herein. For
example, dissociation may be achieved by raising the pH to about
7.0; or, in embodiments in which animal protein free purification
is not necessary, treating the complex with red blood cells at a pH
of about 7.3.
[0054] In a preferred embodiment and to provide animal free toxin,
the complex is subjected to a separation process based on
adjustment pH of the complex in a suitable buffer Suitable buffers
include, but are not limited to, cationic buffers, preferably
cationic buffers that will not interact or will not substantially
interact with the anion exchange column. Suitable cationic buffers
include, e.g., Tris, bis-Tris, triethanolamine, N-methyl
diethanolamine. A pH of between about 7 to about 8.4; more
preferably between about 7.4 to about 8.2; and most preferably a pH
of about 7.8 is typically suitable for dissociating the complex to
release the non-complexed botulinum toxin. In some particularly
preferred embodiments, for example, the pH of the eluent of the
hydrophobic interaction column is raised to about 7.5, about 7.8,
or preferably to about 8.0. For example, in some embodiments, the
eluent may be diluted into a Tris buffer having a pH of about 7.8
to cause the complex to dissociate into individual components,
including the about 150 kD non-complexed botulinum toxin protein.
The resulting mixture comprising dissociated components can then be
subjected to one or more additional chromatography purification
steps, such as ion exchange chromatography steps designed to
capture and further purify the non-complexed toxin.
[0055] In certain embodiments according to the invention, the
non-complexed botulinum toxin may be purified using one or more ion
exchange chromatography steps, (e.g., see FIG. 1A). Ion exchange
chromatography achieves fractionation based on electrostatic
charge. The extent to which a given protein binds to the column
matrix is a function of the protein's net charge, based on its
individual amino acid composition and the charge of the column
matrix. Cationic ion exchange columns have net positive charged
matrix whereas anionic ion exchange columns have a net negative
charged matrix. Bound proteins can be selectively eluted from the
column using a solvent (the eluant) containing a charged substance,
such as salt ions, which competes with the charged matrix support
for binding to the charged proteins. Bound proteins can be thus
fractionated on the basis of the strength of their charge.
Alternatively, proteins may be eluted by adjusted the pH which may
alter the net charge of the protein thereby altering its affinity
to the charged matrix.
[0056] According to some preferred embodiments of the invention,
the mixture comprising non-complexed botulinum toxin is loaded onto
an anion exchange column (e.g., see FIG. 1A). Notably, this column
captures the botulinum toxin in non-complexed form, such that the
toxin protein and dissociated non-toxin proteins can be eluted in
separate fractions. The column used may be any anion column known
in the art suitable for separating charged proteins, non-limiting
examples of which include Q Sepharose HP, Q Sepharose Fast Flow, or
Q XL Sepharose, commercially available from GE Healthcare Life
Sciences. In some particularly preferred embodiments, a Q XL
Sepharose column is used. In some embodiments, the method further
comprises conditioning the mixture comprising the non-complexed
botulinum toxin for anion exchange chromatography before loading
onto the column. For example, buffer and pH conditions may be
determined to optimize yield from the particular column used, as
known in the art, based on the teachings provided herein. For
loading and use in the column, e.g., suitable buffers include, but
are not limited to, cationic buffers, preferably cationic buffers
that will not interact or will not substantially interact with the
anion exchange column. Suitable cationic buffers include, e.g.,
Tris, bis-Tris, triethanolamine, N-methyl diethanolamine. For
loading and equibrilating the column, a pH of between about 7.2 to
about 8.6; more preferably between about 7.4 to about 8.2; and most
preferably a pH of about 7.8 may be used.
[0057] For eluting the captured (bound) toxin and other dissociated
components from the anion exchange column, a suitable buffer can be
used, as known in the art. Examples of suitable buffers include,
for example, sodium chloride (NaCl); and potassium chloride (KCl).
In some particularly preferred embodiments, an ascending gradient
of sodium chloride is used. For example, a sodium chloride buffer
having a concentration range from about 0.0 M to about 0.4 M NaCl,
more preferably from about 0.0 M to about 0.5 M NaCl, and even more
preferably about 0.0 M to about 0.6 M NaCl may be used. Impurities
separated in different fractions may include, e.g., one or more
non-toxin proteins of the dissociated complex, such as, the
non-toxin hemagglutinin and/or non-toxin non-hemagglutinin
proteins. Fraction(s) containing a product peak can be identified,
as known in the art. The peak may occur, for example, at about 8
mSem to about 22 mSem at a pH between about 7.4 to about 8.4, and
preferably at about 7.8, corresponding to about 0.08 M to about
0.18 M NaCl. Conversely, other impurities may elute at about 30 to
about 45 mSem, corresponding to about 0.25 M to about 0.35 M
NaCl.
[0058] The fraction(s) containing the eluted non-complexed
botulinum toxin can be identified to provide an eluent comprising a
non-complexed botulinum toxin. The peak may be identified by
methods as known in the art, e.g., using HPLC, western blot
analysis, ELISA, non-reduced SDS-PAGE, and the like. SDS-PAGE under
non-reducing conditions, for example, can identify the about 150
kDa toxin band, whereas other impurities will appear at bands
corresponding to smaller molecules. This eluent comprising a
non-complexed form may then be subjected to further chromatographic
purification steps.
[0059] In one particularly preferred embodiment, toxin purity is
assessed by SDS-PAGE. As the skilled artisan will appreciate,
SDS-PAGE analysis can be conducted in the absence or presence of
agents that cleave disulfide bonds present in the protein (i.e.,
non-reducing or reducing conditions, respectively). For example,
with respect to botulinum toxin type A, the mature and active form
of the botulinum toxin type A protein molecule is comprised of two
polypeptide chains of 100 kD and 50 kD, respectively, which are
held together by non-covalent interactions as well as a disulfide
bond. When botulinum toxin type A produced by the inventive process
is assayed using non-reducing conditions, the botulinum toxin type
A protein molecules migrate as a single protein band of
approximately 150 kD and the measured purity is typically greater
than 98%. When the botulinum toxin type A protein amount loaded per
gel lane is held to be within the dynamic range of the
densitometer, then there are few, if any, detectable impurity bands
resulting in a measured purity of 100%. When the type A botulinum
toxin is overloaded such that the main toxin band is above the
dynamic range of the densitometer, then some minor impurity bands
may be detectable (as much as 1-2%).
[0060] However, when the SDS-PAGE analysis of botulinum toxin type
A is conducted under reducing conditions, then the disulfide bond
of the botulinum toxin is cleaved and the botulinum toxin type A
protein migrates as two components having molecular weights of 100
kD and 50 kD, respectively. When the botulinum toxin type A protein
is loaded such that the main species are above the dynamic range of
the densitometer and the SDS-page is run under reducing conditions
then minor impurity species can be more easily detected. For
instance, under these conditions there may be as much as 5% of the
150 kD species present due to incomplete proteolytic processing
during the fermentation and recovery process. Under these
conditions the inventive process yields a toxin product (comprised
of the active, cleaved 100 kD and 50 kD polypeptide chains) that is
typically greater than 90% of total protein and more likely greater
than 95% of total protein. Thus, the reported measured purity of
the toxin depends on the details of the SDS-PAGE method employed,
as described herein. Furthermore, while the foregoing example
concerns botulinum toxin type A, the skilled artisan will
appreciate that the SDS-PAGE analysis described herein can be
readily adapted to assess the purity of other serotypes of
botulinum toxin.
[0061] In certain embodiments, the eluent from the anionic column
comprising non-complexed botulinum toxin is loaded onto a cation
exchange column (see, e.g. FIG. 1A). Notably, this column also
captures the botulinum toxin in non-complexed form, such that the
toxin protein and dissociated non-toxin proteins can be eluted in
separate fractions. The column used may be any cation column known
in the art suitable for separating proteins, non-limiting examples
of which include an SP Sepharose column, including SP Sepharose HP
or SP Sephrose Fast Flow; a Mono S column; or a Source-S column,
such as a Source-30S column, or preferably a Source-15S column,
both commercially available from GE Healthcare Life Sciences. In
some embodiments, the method further comprises conditioning the
eluent from the anionic exchange columns comprising non-complexed
botulinum toxin for cation exchange chromatography before loading
onto the column. In some preferred embodiments, the pH is adjusted
so that the pH of the eluent being loaded on the column allows for
efficient binding of the free toxin to the column. For example, the
pH can be maintained within a range of from about 4 to about 8,
preferably from about 5 to about 7.5, more preferably from about 6
to about 7, and most preferably at about 7. Further, in some
embodiments, the eluent from the anionic column can be treated to
reduce conductivity before loading onto the cation exchange column,
e.g., using a sodium phosphate buffer, a non-limiting example of
which is a sodium phosphate buffer of about 20 mM
NaH.sub.2PO.sub.4. For example, the eluent from the anionic column
may contain as much as about 0.15 M NaCl, so that diluting in an
about 20 mM NaH.sub.2PO.sub.4 buffer reduces conductivity. In some
specific embodiments, conductivity is reduced from about 12 mSem to
about 3.3 mSem. Dilution in buffer, dialysis or other methods known
in the art also may be used to reduce the conductivity.
[0062] For loading and use in the column, e.g., suitable buffers
include, but are not limited to, anionic buffers, preferably
anionic buffers that will not interact or will not substantially
interact with the cationic exchange column. Suitable anionic
buffers include, e.g., as MES, HEPES, and the like, and preferably
sodium phosphate buffer. For loading and equibrilating the column,
a pH of between about 4 to about 8; preferably between about 5 to
about 7.5; more preferably from about 6 to about 7; and most
preferably a pH of about 6.8 to about 7 may be used.
[0063] For eluting the captured (bound) toxin from the cation
exchange column separately from other dissociated non-toxin
proteins and other impurities, a suitable buffer can be used, as
known in the art. In some particularly preferred embodiments, an
ascending gradient of sodium chloride is used. A suitable
concentration range for the sodium chloride gradient may be from
about 0.0 M to about 1 M NaCl. Other salts that may be used
include, e.g., potassium chloride, that may be used at a
concentration gradient of about 0.0 M to about 0.5 M KCl.
Fraction(s) containing a product peak can be identified, as known
in the art. The peak may occur, for example from about 18 to about
25 mSem, corresponding to about 0.3 M to about 0.4 M NaCl, at a pH
of about 6.7. That is, the fraction(s) containing the eluted
non-complexed botulinum toxin can be identified to provide an
eluent from the cationic column comprising non-complexed botulinum
toxin. In particularly preferred embodiments, the eluent from the
cationic column represents a non-complexed botulinum toxin of high
purity, in high yield and having high activity. In contrast, the
process outlined in FIG. 1B provides a 900 kD botulinum toxin type
A complex in the final eluent.
[0064] Purified Non-Complexed Botulinum Toxin Product
[0065] The methods and systems described herein are useful to
provide a non-complexed botulinum toxin of high purity, in high
yield, and having high activity. See Example 1 below. The product
is also readily stabilized and conveniently used for the
preparation of safe pharmaceutical compositions.
[0066] In some preferred embodiments, the purified non-complexed
botulinum toxin is at least about 80% pure, preferably at least
about 90% pure, more preferably at least about 95% pure, even more
preferably at least about 98% pure, and most preferably at least
about 99% pure, or even about 100% pure. "Purified non-complexed
botulinum toxin" refers to a free botulinum toxin protein molecule
that is isolated, or substantially isolated, from other proteins
and impurities, which can otherwise accompany the non-complexed
botulinum toxin as it is obtained from a culture or fermentation
process. A purified non-complexed botulinum toxin that is, for
example, "80% pure" refers to an isolated or substantially isolated
non-complexed botulinum toxin protein wherein the toxin protein
comprises 80% of total protein present as determined by or other
suitable analytical methodology, non-limiting examples of which
include SDS-PAGE, CE, and HPLC. For example, in some preferred
embodiments, the cationic column eluent comprising the
non-complexed botulinum toxin is at least about 99% pure, and
contains less than about 1% of host cell proteins that are not the
approximately 150 kD botulinum toxin originally present.
[0067] In some preferred embodiments, the purified non-complexed
botulinum toxin has an activity of at least about 150 LD.sub.50
units/ng, preferably at least about 180 LD.sub.50 units/ng, more
preferably at least about 200 LD.sub.50 units/ng, even more
preferably at least about 210 LD.sub.50 units/ng, and most
preferably at least about 220 LD.sub.50 units/ng. One unit of
botulinum toxin is defined as the LD.sub.50 upon intraperitoneal
injection into female Swiss Webster mice weighing about 18-20 grams
each. In other words, one unit of botulinum toxin is the amount of
botulinum toxin that kills 50% of a group of female Swiss Webster
mice. "Activity" is used interchangeably herein with related
expressions "biological activity", "potency" and "toxicity" to
described the action of a botulinum toxin.
[0068] In preferred embodiments, the non-complexed botulinum toxins
obtainable by processes and systems described herein demonstrate
biological activity. That is, in preferred embodiments, the
biological activity or toxicity of the product is not lost upon
purification in accordance with preferred embodiments of the
present invention, even though non-toxin proteins natively
associated with the toxin protein are removed during purification.
In even more preferred embodiments, the potency obtained using a
given set of processes and parameters within the scope of the
invention is consistent and/or reproducible. For example, the
potency measurement can be made with less than about 40%
variability, preferably less than about 35% variability, more
preferably less than about 30% variability, even more preferably
less than about 25% variability, and most preferably less than
about 20% variability.
[0069] In some preferred embodiments, the purification process
provides the non-complexed botulinum toxin in high yield. For
example, the yield obtained from 30 L of a fermentation culture may
be at least about 30 mg, preferably at least about 40 mg, more
preferably at least about 70 mg, even more preferably at least
about 80 mg, and most preferably at least about 90 mg,
corresponding to a yield of at least about 1 mg/L, preferably at
least about 1.3 mg/L, more preferably at least about 2.3 mg/L, even
more preferably at least about 2.7 mg/L, and most preferably at
least about 3 mg/L, respectively. In even more preferred
embodiments, the yield obtained using a given set of processes and
parameters within the scope of the invention is reproducible. For
example, yield can be measured with less than about 40%
variability, preferably less than about 35% variability, more
preferably less than about 30% variability, even more preferably
less than about 25% variability, and most preferably less than
about 20% variability.
[0070] In some particularly preferred embodiments, the purified
non-complexed botulinum toxin is stable during purification using
the processes and systems described herein. It has been believed
that removal of associated non-toxin proteins from a botulinum
toxin complex, such as botulinum toxin type A complex, results in a
markedly unstable botulinum toxin product. The instant invention,
however, provides methods and systems that can stably isolate free
botulinum toxin, without associated non-toxin proteins
conventionally believed necessary during the purification process
to maintain stability, as discussed above.
[0071] In some preferred embodiments, methods and systems described
herein provide a non-complexed botulinum toxin that requires very
few post-chromatography steps, e.g., in terms of maintaining
stability during storage, and in terms of applicability to
pharmaceutical uses. For example, as known in the art, ammonium
sulfate may be added to the free botulinum toxin to prepare an
ammonium sulfate suspension for storage. The composition comprising
free botulinum toxin and ammonium sulfate may be readily stored in
a refrigerator and later can be readily retrieved for use in
pharmaceutical applications. Indeed, the stability, high yield and
purity, and high and consistent potency of the toxin obtainable by
methods described herein facilitate pharmaceutical use of the
purified product, as described in more detail below.
[0072] Uses of Purified Non-Complexed Botulinum Toxin
[0073] The non-complexed botulinum toxin purified according to this
invention can be used in the preparation of pharmaceutical
compositions comprising the toxin as an active ingredient for
administration to any subject who would receive a benefit from such
pharmaceutical compositions. In preferred embodiments, the subjects
to be treated are mammals, preferably humans. "Pharmaceutical
composition" as used herein refers to a formulation in which an
active ingredient can be a botulinum toxin. The formulation will
contain at least one additional ingredient and be suitable for
diagnostic, therapeutic, and/or or cosmetic administration to a
subject, such as a human patient. The pharmaceutical composition
can be liquid or solid; and may be a single or multi-component
system, for example a lyophilized composition reconstituted with a
diluent such as saline.
[0074] Another aspect of the invention provides for administration
of a purified botulinum toxin molecule to a patient.
"Administration" as used herein refers to providing a
pharmaceutical composition to a subject or patient. The
pharmaceutical composition may be administered by, any method known
in the art, including e.g., intramuscular (i.m.), intradermal,
intranasal, or subcutaneous administration, intrathecal
administration, intracranial, intraperitoneal (i.p.)
administration, or topical (transdermal) and implantation (e.g., of
a slow-release device) routes of administration. In certain
preferred embodiments, the purified non-complexed botulinum toxin
is administered topically or by injection in compositions as
described in U.S. patent application Ser. Nos. 09/910,432;
10/793,138; 11/072,026; 11/073,307, 11/824,393, and 12/154,982,
which are hereby incorporated by reference in their entirety.
[0075] In certain embodiments, compositions comprising
non-complexed botulinum toxin in an ammonium sulfate suspension can
be readily compounded into a pharmaceutical composition. For
example, an ammonium sulfate suspension comprising non-complexed
botulinum toxin protein can be centrifuged to recover the protein
and the protein can be re-solubilized, diluted, and compounded with
one or more pharmaceutically acceptable excipients. In certain
embodiments, the pharmaceutical composition may comprise a
non-complexed botulinum toxin as an active pharmaceutical
ingredient, and may further comprise one or more buffers, carriers,
stabilizers, preservatives and/or bulking agents. The
pharmaceutical compositions may be lyophilized to powder for
storage, and re-constituted for further use. Accordingly, processes
and systems described herein can provide a botulinum toxin in a
form particularly suited to pharmaceutical applications terms of
ease of preparation.
[0076] The pharmaceutical composition may find use in therapeutic,
diagnostic, research and/or cosmetic applications. For example, as
discussed above, botulinum toxin type A is clinically used to treat
neuromuscular disorders characterized by skeletal muscle
hyperactivity, such as essential blepharospasm, strabismus,
cervical dystonia, and glabellar line (facial) wrinkles. Moreover,
in certain applications, non-complexed (about 150 kD) botulinum
toxin is the preferred form for treating humans. See, e.g., Kohl
A., et al., Comparison of the effect of botulinum toxin A
(Botox.RTM.) with the highly-purified neurotoxin (NT 201) in the
extensor digitorum brevis muscle test, Mov Disord 2000; 15(Suppl
3):165. Accordingly, certain botulinum toxin pharmaceutical
compositions are preferably prepared using non-complexed botulinum
toxin, as opposed to a botulinum toxin complex.
EXAMPLES
Example 1
Comparison of Inventive Process with a Modified Schantz Process
[0077] Purifications of non-complexed botulinum toxin type A using
processes within the scope of the instant invention (`inventive
process") were directly compared to purifications based on the
traditional Schantz approach, further modified by the addition of
chromatographic steps to provide the non-complexed form (Modified
Schantz process"). Briefly, Clostridium botulinum bacteria were
cultured and allowed to grow until fermentation was complete
(usually about 72 to about 120 hours from inoculation to harvest).
A volume of 30 L of the fermentation culture then was used in each
of the following purification procedures.
[0078] The modified Schantz process used involved typical
acidification of the fermentation culture to precipitate the toxin,
followed by ultramicrofiltration (UF) and diafiltration (DF) to
concentrate the raw toxin. DNase and RNase were added to the
harvested toxin to digest (hydrolyze) nucleic acids, which were
then removed by an additional UF step, using tangential flow
filtration (300 kD UF). The toxin was next extracted with phosphate
buffer, followed by three sequential precipitation steps: cold
ethanol precipitation; hydrochloric acid precipitation, and
ammonium sulfate precipitation, where the supernatants each time
were normally discarded. This procedure provided a 900 kD botulinum
toxin type A complex, which was then subjected to additional
chromatography steps to provide the free toxin. Specifically, the
toxin complex was resolubilized and subjected to negative batch
adsorption onto a DEAE resin. The eluent was then run on a gravity
flow anion exchange column (DEAE-Sepharose), followed by a gravity
flow cation exchange column (CM-Sepharose). Yield was determined,
the length of time the process took was recorded (not counting the
fermentation period), and the purified non-complexed botulinum
toxin type A was measured for purity by SDS-PAGE analysis and
assayed for potency, e.g., by techniques known to those skilled in
the art. The entire modified Schantz process was repeated for three
different lots, lot numbers 1, 2 and 3, and the results recorded in
Table 1 below.
[0079] The inventive process was used with three different lots,
lot numbers 4, and 6, in accordance with systems and methods
described herein. Briefly, the fermentation culture was subjected
to acid precipitation using 3M sulfuric acid to reduce pH to 3.5,
at a temperature below 25.degree. C. The acid precipitate was then
subjected to 0.1 .mu.m tangential flow filtration to concentrate
cell mass. The pH then was adjusted to 6 and nucleases added to
reduce host cell nucleic acid content, followed by clarification by
centrifugation to remove cell debris and dead end filtration at 0.2
.mu.m with added ammonium sulfate. The filtrate was then directly
loaded onto the hydrophobic interaction column, Phenyl Sepharose HP
(GE Life Sciences), eluted with a descending gradient of ammonium
sulfate, and the product peak isolated. The eluent was then diluted
into Tris buffer pH 7.8 to dissociate the toxin complex, which then
was loaded onto the anion exchange column Q XL Sepharose (GE
Lifesciences), eluted with an ascending gradient of sodium
chloride, and again the product peak collected. This eluent was
then diluted in a sodium phosphate buffer (to reduce conductivity)
and loaded onto either the anion exchange column, Q XL Sepharose
(for lots #4 and 5), or the cation exchange column, Source-S (GE
Life Sciences) (for lot #6), again eluted with an ascending
gradient of sodium chloride, and a final product peak collected and
stored. This process yielded non-complexed botulinum toxin type A.
Yield was determined, the length of process time recorded (not
counting the fermentation period), and the toxin measured for
purity by SDS-PAGE analysis and assayed for potency, e.g., by
techniques known to those skilled in the art. Results also recorded
Table 1 below.
TABLE-US-00001 TABLE 1 Modified Schantz Process Inventive Process
Lot # 1 2 3 4 5 6 Process 10 days 4 days Time % Purity 99 n/a 97
98.6 95.3 100 Yield (30 L 11 mg 0 mg 4 mg 43 mg 99 mg 89 mg scale)
Potency 255 n/a 173 259 252 250 (LD50 Units/ng)
[0080] As Table 1 indicates, there was a lot failure with respect
to lot #2 in the modified Schantz process. The total lot failed
giving zero yield. There was also a partial lot failure with
respect to lot #3. There the failure occurred at the hydrochloric
acid precipitation step, but some product was rescued from the
normally discarded supernatant. The rescued product was reprocessed
with a deviation step, accounting for the observed reduced yield
compared with lot #1 (4 mg compared with 11 mg) and the observed
reduced potency compared with lot #1 (173 LD50 units/ng compared
with 255 LD50 units/ng).
[0081] With respect to the lots used with the inventive process,
lot #4 showed a reduced yield, compared to lot #5 for example (43
mg compared with 99 mg) due to a chromatography system failure,
involving high salt wash of a column. With the failure, there was
premature elution of a portion of the toxin, resulting in the
observed reduced yield, but also an observed higher purity (98.6%
purity compared with 95.3% purity).
[0082] Lot #6 represents the results of a highly preferred
embodiment of the instant inventive processes and systems, where a
cation exchange column was used in the third chromatography step.
As Table 1 indicates, this embodiment resulted in improved purity
compared with lot #5 for example (100% purity compared with 95.3%
purity), while high yield (89 mg compared with 99 mg) and high
potency (250 LD50 units/ng compared with 252 LD50 units/ng) were
maintained.
[0083] As Table 1 also indicates, the total length of the
purification can be shortened in preferred embodiments of the
instant invention. For example, lot #6 was purified within only 4
days, compared to the 10 days it took to purify non-complexed
botulinum toxin using the modified Schantz method that involved
three additional chromatography steps after the conventional
Schantz method.
[0084] The results indicate that the processes and systems taught
herein can be used to prepare high yields of a non-complexed
botulinum toxin, at high potency and purity, and suggests that
methods and systems described herein can find use in large-scale
efficient purification of a non-complexed botulinum toxin suitable
for use, e.g., as an active ingredient in pharmaceutical
compositions.
[0085] All references including patent applications and
publications cited herein are incorporated herein by reference in
their entirety and for all purposes to the same extent as if each
individual publication or patent or patent application was
specifically and individually indicated to be incorporated by
reference in its entirety for all purposes. Many modifications and
variations of this invention can be made without departing from its
spirit and scope, as will be apparent to those skilled in the art.
The specific embodiments described herein are offered by way of
example only, and the invention is to be limited only by the terms
of the appended claims, along with the full scope of equivalents to
which such claims are entitled.
* * * * *