U.S. patent application number 11/932789 was filed with the patent office on 2009-05-14 for chromatographic method and system for purifying a botulinum toxin.
This patent application is currently assigned to ALLERGAN, INC.. Invention is credited to Stephen Donovan, Mingjiang Luo, Hui Xiang, Yao Yu.
Application Number | 20090123497 11/932789 |
Document ID | / |
Family ID | 46205966 |
Filed Date | 2009-05-14 |
United States Patent
Application |
20090123497 |
Kind Code |
A1 |
Luo; Mingjiang ; et
al. |
May 14, 2009 |
CHROMATOGRAPHIC METHOD AND SYSTEM FOR PURIFYING A BOTULINUM
TOXIN
Abstract
Chromatographic processes and systems for purifying a botulinum
toxin from an APF fermentation medium.
Inventors: |
Luo; Mingjiang; (Newport
Beach, CA) ; Xiang; Hui; (Irvine, CA) ; Yu;
Yao; (Yorba Linda, CA) ; Donovan; Stephen;
(Capistrano Beach, CA) |
Correspondence
Address: |
ALLERGAN, INC.
2525 DUPONT DRIVE, T2-7H
IRVINE
CA
92612-1599
US
|
Assignee: |
ALLERGAN, INC.
Irvine
CA
|
Family ID: |
46205966 |
Appl. No.: |
11/932789 |
Filed: |
October 31, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11452570 |
Jun 14, 2006 |
7452697 |
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11932789 |
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11072673 |
Mar 3, 2005 |
7354740 |
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11452570 |
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11072050 |
Mar 3, 2005 |
7160699 |
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11072673 |
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10672876 |
Sep 25, 2003 |
7148041 |
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11072050 |
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Current U.S.
Class: |
424/239.1 ;
435/252.7; 435/71.3 |
Current CPC
Class: |
A61P 25/00 20180101;
C12N 1/20 20130101; C12N 9/6489 20130101; C12Y 304/24069 20130101;
C12P 21/02 20130101; C12N 9/52 20130101; C07K 14/33 20130101 |
Class at
Publication: |
424/239.1 ;
435/71.3; 435/252.7 |
International
Class: |
A61K 39/08 20060101
A61K039/08; C12P 21/00 20060101 C12P021/00; A61P 25/00 20060101
A61P025/00; C12N 1/20 20060101 C12N001/20 |
Claims
1. A method for obtaining a biologically active botulinum toxin,
comprising the steps of: (a) providing a fermentation medium that
is substantially free of an animal derived product; (b) culturing a
Clostridium botulinum bacterium in the fermentation medium under
conditions which permit production of a botulinum toxin, and; (c)
recovering a biologically active botulinum toxin from the
fermentation medium.
2. The method of claim 1, wherein in the step of providing a
fermentation medium, the medium comprises a protein product derived
from a vegetable.
3. The method of claim 2, wherein the vegetable is a soybean.
4. The method of claim 1, wherein in the step of culturing, the
culturing is performed until cell density of the culture decreases
due to cell lysis.
5. The method of claim 1, wherein in the step of culturing, the
culturing is performed until at least 48 hours after initial drop
in cell density due to cell lysis.
6. A method for production of a botulinum toxin, the method
comprising steps of: (a) providing a first medium that is
substantially free of an animal derived product and that comprises
a protein product derived from a vegetable; (b) culturing a
Clostridium botulinum bacterium the first medium under conditions
that permit growth of the Clostridium botulinum; (c) providing a
second medium that is substantially free of an animal derived
product and that comprises a protein product derived from a
vegetable; (d) inoculating the second medium with the first medium;
(e) culturing a Clostridium botulinum bacterium in the second
medium under conditions that allow production of a botulinum toxin,
and; (f) recovering the botulinum toxin.
7. The method of claim 6, wherein in the step of providing a first
medium, the first medium comprises a protein product derived from a
soybean, and wherein in the step of inoculating the second medium,
the second medium comprises the protein product derived from a
soybean.
8. The method of claim 6, wherein in the step of providing a first
medium, the first medium comprises a hydrolyzed soy, and wherein in
the step of inoculating a second medium, the fermentation medium
comprises hydrolyzed soy.
9. The method of claim 6, wherein in the step of culturing a
Clostridium botulinum in a first medium, the conditions comprise a
temperature of about 34 degrees Celsius, and further comprise no
decrease in cell density during culturing, wherein in the step of
inoculating a second medium with a first medium, 2 to 4 percent of
the first medium is used to inoculate the second medium, and
wherein in the step of culturing the bacterium in the second
medium, the conditions that allow growth comprise a temperature of
approximately 34 degrees Celsius and further comprise culturing
until cell density of the culture decreases due to cell lysis.
10. A composition comprising a Clostridium botulinum and a culture
medium for producing a botulinum toxin wherein the medium is
substantially free of an animal derived product and comprises a
protein product derived from a vegetable.
11. The composition of claim 10, wherein the vegetable is a
soybean.
12. The composition of claim 10, wherein the composition comprises
a hydrolyzed soy.
13. A method for making a substantially animal product free
pharmaceutical composition in which the active ingredient is a
botulinum toxin, the method comprising the steps of: (a) obtaining
a biologically active botulinum toxin by; (i) providing a
fermentation medium that is substantially free of an animal derived
product; (ii) culturing a Clostridium botulinum in the fermentation
medium under conditions which permit production of a botulinum
toxin, and; (iii) recovering a biologically active botulinum toxin
from the fermentation medium; (b) formulating the botulinum toxin
with a suitable excipient, thereby making a substantially animal
product free pharmaceutical composition in which the active
ingredient is a botulinum toxin.
Description
CROSS REFERENCE
[0001] This application is a continuation of U.S. patent
application Ser. No. 11/452,570, filed Jun. 14, 2006, which is a
continuation in part of U.S. patent application Ser. No.
11/072,673, filed Mar. 3, 2005, which is a continuation in part of
application Ser. No. 11/072,050, filed Mar. 3, 2005, which is a
continuation in part of U.S. application Ser. No. 10/672,876, filed
Sep. 25, 2003, the entire contents of which applications are
incorporated herein by reference.
BACKGROUND
[0002] The present invention relates to systems and processes for
purifying a Clostridium toxin. In particular, the present invention
relates to a chromatographic process for purifying a botulinum
neurotoxin. A pharmaceutical composition suitable for
administration to a human or animal for a therapeutic, diagnostic,
research or cosmetic purpose can comprise an active ingredient. The
pharmaceutical composition can also include one or more excipients,
buffers, carriers, stabilizers, preservatives and/or bulking
agents. The active ingredient in a pharmaceutical composition can
be a biologic such as a botulinum toxin. The botulinum toxin active
ingredient used to make a botulinum toxin pharmaceutical
composition can be obtained through a multi step culturing,
fermentation and compounding process which makes use of one or more
animal derived products (such as meat broth and casein ingredients
in one or more of the culture and fermentation media used to obtain
a bulk botulinum toxin, and a blood fraction or blood derivative
excipient in the final compounded botulinum toxin pharmaceutical
composition). Administration to a patient of a pharmaceutical
composition wherein the active ingredient biologic is obtained
through a process which makes use of animal derived products can
subject the patient to a potential risk of receiving various
pathogens or infectious agents. For example, prions may be present
in a pharmaceutical composition. A prion is a proteinaceous
infectious particle which is hypothesized to arise as an abnormal
conformational isoform from the same nucleic acid sequence which
makes the normal protein. It has been further hypothesized that
infectivity resides in a "recruitment reaction" of the normal
isoform protein to the prion protein isoform at a post
translational level. Apparently, the normal endogenous cellular
protein is induced to misfold into a pathogenic prion
conformation.
[0003] Creutzfeldt-Jacob disease is a rare neurodegenerative
disorder of human transmissible spongiform encephalopathy where the
transmissible agent is apparently an abnormal isoform of a prion
protein. An individual with Creutzfeldt-Jacob disease can
deteriorate from apparent perfect health to akinetic mutism within
six months. Thus, a potential risk may exist of acquiring a prion
mediated disease, such as Creutzfeldt-Jacob disease, from the
administration of a pharmaceutical composition which contains a
biologic, such as a botulinum toxin, obtained, purified or
compounded using animal derived products.
Botulinum Toxin
[0004] The genus Clostridium has more than one hundred and twenty
seven species, grouped by morphology and function. The anaerobic,
gram positive bacterium Clostridium botulinum produces a potent
polypeptide neurotoxin, botulinum toxin (synonymously "toxin"),
which causes a neuroparalytic illness in humans and animals known
as botulism. Clostridium botulinum and its spores are commonly
found in soil and the bacterium can grow in improperly sterilized
and sealed food containers of home based canneries, which are the
cause of many of the cases of botulism. The effects of botulism
typically appear 18 to 36 hours after eating the foodstuffs
infected with a Clostridium botulinum culture or spores. The
botulinum toxin can apparently pass unattenuated through the lining
of the gut and attack peripheral motor neurons. Symptoms of
botulinum toxin intoxication can progress from difficulty walking,
swallowing, and speaking to paralysis of the respiratory muscles
and death.
[0005] Botulinum toxin type A is the most lethal natural biological
agent known to man. About 50 picograms of botulinum toxin (purified
neurotoxin complex) type A is a LD.sub.50 in mice. On a molar
basis, botulinum toxin type A is 1.8 billion times more lethal than
diphtheria, 600 million times more lethal than sodium cyanide, 30
million times more lethal than cobrotoxin and 12 million times more
lethal than cholera. Singh, Critical Aspects of Bacterial Protein
Toxins, pages 63-84 (chapter 4) of Natural Toxins II, edited by B.
R. Singh et al., Plenum Press, New York (1976) (where the stated
LD.sub.50 of botulinum toxin type A of 0.3 ng equals 1 U is
corrected for the fact that about 0.05 ng of BOTOX.RTM. equals 1
unit). BOTOX.RTM. is the trademark of a botulinum toxin type A
purified neurotoxin complex available commercially from Allergan,
Inc., of Irvine, Calif. One unit (U) 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. Seven generally
immunologically distinct botulinum neurotoxins have been
characterized, these being respectively botulinum neurotoxin
serotypes A, B, C.sub.1, D, E, F and G each of which is
distinguished by neutralization with type-specific antibodies. The
different serotypes of botulinum toxin vary in the animal species
that they affect and in the severity and duration of the paralysis
they evoke. For example, it has been determined that botulinum
toxin type A is 500 times more potent, as measured by the rate of
paralysis produced in the rat, than is botulinum toxin type B.
Additionally, botulinum toxin type B has been determined to be
non-toxic in primates at a dose of 480 U/kg which is about 12 times
the primate LD.sub.50 for botulinum toxin type A. The botulinum
toxins apparently bind with high affinity to cholinergic motor
neurons, are translocated into the neuron and block the presynaptic
release of acetylcholine.
[0006] Botulinum toxins have been used in clinical settings for the
treatment of e.g. neuromuscular disorders characterized by
hyperactive skeletal muscles. Botulinum toxin type A has been
approved by the U.S. Food and Drug Administration for the treatment
of essential blepharospasm, strabismus and hemifacial spasm in
patients over the age of twelve, for the treatment of cervical
dystonia and for the treatment of glabellar line (facial) wrinkles.
The FDA has also approved a botulinum toxin type B for the
treatment of cervical dystonia. Clinical effects of peripheral
injection (i.e. intramuscular or subcutaneous) botulinum toxin type
A are usually seen within one week of injection, and often within a
few hours after injection. The typical duration of symptomatic
relief (i.e. flaccid muscle paralysis) from a single intramuscular
injection of botulinum toxin type A can be about three months to
about six months.
[0007] Although all the botulinum toxins serotypes apparently
inhibit release of the neurotransmitter acetylcholine at the
neuromuscular junction, they do so by affecting different
neurosecretory proteins and/or cleaving these proteins at different
sites. Botulinum toxin A is a zinc endopeptidase which can
specifically hydrolyze a peptide linkage of the intracellular,
vesicle associated protein SNAP-25. Botulinum type E also cleaves
the 25 kiloDalton (kD) synaptosomal associated protein (SNAP-25),
but targets different amino acid sequences within this protein, as
compared to botulinum toxin type A. Botulinum toxin types B, D, F
and G act on vesicle-associated protein (VAMP, also called
synaptobrevin), with each serotype cleaving the protein at a
different site. Finally, botulinum toxin type C.sub.1 has been
shown to cleave both syntaxin and SNAP-25. These differences in
mechanism of action may affect the relative potency and/or duration
of action of the various botulinum toxin serotypes.
[0008] Regardless of serotype, the molecular mechanism of toxin
intoxication appears to be similar and to involve at least three
steps or stages. In the first step of the process, the toxin binds
to the presynaptic membrane of the target neuron through a specific
interaction between the heavy chain (H chain) and a cell surface
receptor; the receptor is thought to be different for each serotype
of botulinum toxin. The carboxyl end segment of the H chain,
H.sub.C, appears to be important for targeting of the toxin to the
cell surface.
[0009] In the second step, the toxin crosses the plasma membrane of
the poisoned cell. The toxin is first engulfed by the cell through
receptor-mediated endocytosis, and an endosome containing the toxin
is formed. The toxin then escapes the endosome into the cytoplasm
of the cell. This last step is thought to be mediated by the amino
end segment of the H chain, H.sub.N, which triggers a
conformational change of the toxin in response to a pH of about 5.5
or lower. Endosomes are known to possess a proton pump which
decreases intra endosomal pH. The conformational shift exposes
hydrophobic residues in the toxin, which permits the toxin to embed
itself in the endosomal membrane. The toxin then translocates
through the endosomal membrane into the cytosol.
[0010] The last step of the mechanism of botulinum toxin activity
appears to involve reduction of the disulfide bond joining the H
and L chain. The entire toxic activity of botulinum and botulinum
toxins is contained in the L chain of the holotoxin; the L chain is
a zinc (Zn.sup.2+) endopeptidase which selectively cleaves proteins
essential for recognition and docking of
neurotransmitter-containing vesicles with the cytoplasmic surface
of the plasma membrane, and fusion of the vesicles with the plasma
membrane. Botulinum neurotoxin, botulinum toxin B, D, F, and G
cause degradation of synaptobrevin (also called vesicle-associated
membrane protein (VAMP)), a synaptosomal membrane protein. Most of
the VAMP present at the cytosolic surface of the synaptic vesicle
is removed as a result of any one of these cleavage events. Each
toxin specifically cleaves a different bond.
[0011] The molecular weight of the botulinum toxin protein
molecule, for all seven of the known botulinum toxin serotypes, is
about 150 kD. Interestingly, the botulinum toxins are released by
Clostridial bacterium as complexes comprising the 150 kD botulinum
toxin protein molecule along with one or more associated non-toxin
proteins. Thus, the botulinum toxin type A complex can be produced
by Clostridial bacterium as 900 kD, 500 kD and 300 kD forms
(approximate molecular weights). Botulinum toxin types B and
C.sub.1 are apparently produced as only a 500 kD complex. Botulinum
toxin type D is produced as both 300 kD and 500 kD complexes.
Finally, botulinum toxin types E and F are produced as only
approximately 300 kD complexes. The complexes (i.e. molecular
weight greater than about 150 kD) are believed to contain a
non-toxin hemagglutinin protein and a non-toxin and non-toxic
nonhemagglutin in protein. Thus, a botulinum toxin complex can
comprise a botulinum toxin molecule (the neurotoxic component) and
one or more non toxic, hemagluttinin proteins and/or non toxin, non
hemagluttinin proteins (the later can be referred to as NTNH
proteins). These two types of non-toxin proteins (which along with
the botulinum toxin molecule can comprise the relevant neurotoxin
complex) may act to provide stability against denaturation to the
botulinum toxin molecule and protection against digestive acids
when toxin is ingested. Additionally, it is possible that the
larger (greater than about 150 kD molecular weight) botulinum toxin
complexes may result in a slower rate of diffusion of the botulinum
toxin away from a site of intramuscular injection of a botulinum
toxin complex. The toxin complexes can be dissociated into toxin
protein and hemagglutinin proteins by treating the complex with red
blood cells at pH 7.3 or by subjecting the complex to a separation
process, such as column chromatography, in a suitable buffer at a
pH of about 7-8. The botulinum toxin protein has A marked
instability upon removal of the hemagglutinin protein.
[0012] All the botulinum toxin serotypes are made by native
Clostridium botulinum bacteria as inactive single chain proteins
which must be cleaved or nicked by proteases to become neuroactive.
The bacterial strains that make botulinum toxin serotypes A and G
possess endogenous proteases and serotypes A and G can therefore be
recovered from bacterial cultures in predominantly their active
form. In contrast, botulinum toxin serotypes C.sub.1, D, and E are
synthesized by nonproteolytic strains and are therefore typically
unactivated when recovered from culture. Serotypes B and F are
produced by both proteolytic and nonproteolytic strains and
therefore can be recovered in either the active or inactive form.
However, even the proteolytic strains that produce, for example,
the botulinum toxin type B serotype only cleave a portion of the
toxin produced. The exact proportion of nicked to unnicked
molecules depends on the length of incubation and the temperature
of the culture. Therefore, a certain percentage of any preparation
of, for example, the botulinum toxin type B toxin is likely to be
inactive, possibly accounting for the known significantly lower
potency of botulinum toxin type B as compared to botulinum toxin
type A. The presence of inactive botulinum toxin molecules in a
clinical preparation will contribute to the overall protein load of
the preparation, which has been linked to increased antigenicity,
without contributing to its clinical efficacy. Additionally, it is
known that botulinum toxin type B has, upon intramuscular
injection, a shorter duration of activity and is also less potent
than botulinum toxin type A at the same dose level.
[0013] In vitro studies have indicated that botulinum toxin
inhibits potassium cation induced release of both acetylcholine and
norepinephrine from primary cell cultures of brainstem tissue.
Additionally, it has been reported that botulinum toxin inhibits
the evoked release of both glycine and glutamate in primary
cultures of spinal cord neurons and that in brain synaptosome
preparations botulinum toxin inhibits the release of each of the
neurotransmitters acetylcholine, dopamine, norepinephrine, CGRP and
glutamate.
[0014] The success of botulinum toxin type A to treat a variety of
clinical conditions has led to interest in other botulinum toxin
serotypes. Thus, at least botulinum toxins types, A, B, E and F
have been used clinically in humans. Additionally, pure (approx 150
kDa) botulinum toxin has been used to treat humans. See e.g. Kohl
A., et al., Comparison of the effect of botulinum toxin A (Botox
(R)) with the highly-purified neurotoxin (NT 201) in the extensor
digitorum brevis muscle test, Mov Disord 2000; 15(Suppl 3):165.
Hence, a botulinum toxin pharmaceutical composition can be prepared
using a pure (approx 150 kDa) botulinum toxin, as opposed to use of
a botulinum toxin complex.
[0015] The type A botulinum toxin is known to be soluble in dilute
aqueous solutions at pH 4-6.8. At pH above about 7 the stabilizing
nontoxic proteins dissociate from the neurotoxin, resulting in a
gradual loss of toxicity, particularly as the pH and temperature
rise. Schantz E. J., et al Preparation and characterization of
botulinum toxin type A for human treatment (in particular pages
44-45), being chapter 3 of Jankovic, J., et al, Therapy with
Botulinum Toxin, Marcel Dekker, Inc (1994).
[0016] As with enzymes generally, the biological activities of the
botulinum toxins (which are intracellular peptidases) is dependant,
at least in part, upon their three dimensional conformation. Thus,
botulinum toxin type A is detoxified by heat, various chemicals
surface stretching and surface drying. Additionally, it is known
that dilution of the toxin complex obtained by the known culturing,
fermentation and purification to the much, much lower toxin
concentrations used for pharmaceutical composition formulation
results in rapid detoxification of the toxin unless a suitable
stabilizing agent is present. Dilution of the toxin from milligram
quantities to a solution containing nanograms per milliliter
presents significant difficulties because of the rapid loss of
specific toxicity upon such great dilution. Since the toxin may be
used months or years after the toxin containing pharmaceutical
composition is formulated, the toxin can be stabilized with a
stabilizing agent such as albumin and gelatin.
[0017] It has been reported that a botulinum toxin has been used in
various clinical settings, including as follows:
(1) about 75-125 units of BOTOX.RTM. per intramuscular injection
(multiple muscles) to treat cervical dystonia; (2) 5-10 units of
BOTOX.RTM. per intramuscular injection to treat glabellar lines
(brow furrows) (5 units injected intramuscularly into the procerus
muscle and 10 units injected intramuscularly into each corrugator
supercilii muscle); (3) about 30-80 units of BOTOX.RTM. to treat
constipation by intrasphincter injection of the puborectalis
muscle; (4) about 1-5 units per muscle of intramuscularly injected
BOTOX.RTM. to treat blepharospasm by injecting the lateral
pre-tarsal orbicularis oculi muscle of the upper lid and the
lateral pre-tarsal orbicularis oculi of the lower lid. (5) to treat
strabismus, extraocular muscles have been injected intramuscularly
with between about 1-5 units of BOTOX.RTM., the amount injected
varying based upon both the size of the muscle to be injected and
the extent of muscle paralysis desired (i.e. amount of diopter
correction desired). (6) to treat upper limb spasticity following
stroke by intramuscular injections of BOTOX.RTM. into five
different upper limb flexor muscles, as follows: [0018] (a) flexor
digitorum profundus: 7.5 U to 30 U [0019] (b) flexor digitorum
sublimus: 7.5 U to 30 U [0020] (c) flexor carpi ulnaris: 10 U to 40
U [0021] (d) flexor carpi radialis: 15 U to 60 U [0022] (e) biceps
brachii: 50 U to 200 U. Each of the five indicated muscles has been
injected at the same treatment session, so that the patient
receives from 90 U to 360 U of upper limb flexor muscle BOTOX.RTM.
by intramuscular injection at each treatment session. (7) to treat
migraine, pericranial injected (injected symmetrically into
glabellar, frontalis and temporalis muscles) injection of 25 U of
BOTOX.RTM. has showed significant benefit as a prophylactic
treatment of migraine compared to vehicle as measured by decreased
measures of migraine frequency, maximal severity, associated
vomiting and acute medication use over the three month period
following the 25 U injection.
[0023] It is known that botulinum toxin type A can have an efficacy
for up to 12 months (European J. Neurology 6 (Supp 4):
S111-S1150:1999), and in some circumstances for as long as 27
months. The Laryngoscope 109:1344-1346:1999. However, the usual
duration of an intramuscular injection of Botox.RTM. is typically
about 3 to 4 months.
[0024] A commercially available botulinum toxin containing
pharmaceutical composition is sold under the trademark BOTOX.RTM.
(available from Allergan, Inc., of Irvine, Calif.). BOTOX.RTM.
consists of a purified botulinum toxin type A complex, human serum
albumin, and sodium chloride packaged in sterile, vacuum-dried
form. The botulinum toxin type A is made from a culture of the Hall
strain of Clostridium botulinum grown in a medium containing N-Z
amine casein and yeast extract. The botulinum toxin type A complex
is purified from the culture solution by a series of acid or acid
and ethanol precipitations to a crystalline complex consisting of
the active high molecular weight toxin protein and an associated
hemagglutinin protein. The crystalline complex is re-dissolved in a
solution containing saline and albumin and sterile filtered (0.2
microns) prior to vacuum-drying. BOTOX.RTM. can be reconstituted
with sterile, non-preserved saline prior to intramuscular
injection. Each vial of BOTOX.RTM. contains about 100 units (U) of
Clostridium botulinum toxin type A complex, 0.5 milligrams of human
serum albumin and 0.9 milligrams of sodium chloride in a sterile,
vacuum-dried form without a preservative.
[0025] To reconstitute vacuum-dried BOTOX.RTM. sterile normal
saline without a preservative (0.9% Sodium Chloride injection) is
used by drawing up the proper amount of diluent in the appropriate
size syringe. Since BOTOX.RTM. is denatured by bubbling or similar
violent agitation, the diluent is gently injected into the vial.
Reconstituted BOTOX.RTM. can be stored in a refrigerator (2.degree.
to 8.degree. C.) and is a clear, colorless liquid and free of
particulate matter. There are reports of reconstituted BOTOX.RTM.
retaining its potency for up to thirty days. See e.g. Guttman C.,
Botox retains its efficacy for blepharospasm treatment after
freezing and storage, New York investigators find, EuroTimes 2000
Nov/Dec;5(8):16. The vacuum-dried product is stored in a freezer at
or below -5.degree. C.
[0026] In general, four physiologic groups of C. botulinum are
recognized (I, II, III, IV). The organisms capable of producing a
serologically distinct toxin may come from more than one
physiological group. For example, Type B and F toxins can be
produced by strains from Group I or II. In addition, other strain
of clostridial species (C. baratii, type F; C. butyricum, type E;
C. novyi, type C.sub.1 or D) have been identified which can produce
botulinum neurotoxins.
[0027] The physiologic groups of Clostridium botulinum types are
listed in Table I.
TABLE-US-00001 TABLE I Physiologic Groups of Clostridium botulinum
Phenotypically Toxin Phages Related Sero- Milk Glucose &
Clostridium Group Type Biochemistry Digest Fermentation Lipase
Plasmids (nontoxigenic) I A, B, F proteolytic saccharolytic + + + +
C. sporogenes II B, E, F nonproteolytic saccharolytic - + + +
psychotrophic III C, D Nonproteolytic saccharolytic .+-. + + + C.
novyi IV G proteolytic nonsaccharolytic + - - - C. subterminale
[0028] These toxin types may be produced by selection from the
appropriate physiologic group of Clostridium botulinum organisms.
The organisms designated as Group I are usually referred to as
proteolytic and produce botulinum toxins of types A, B and F. The
organisms designated as Group II are saccharolytic and produce
botulinum toxins of types B, E and F. The organisms designated as
Group III produce only botulinum toxin types C and D and are
distinguished from organisms of Groups I and II by the production
of significant amounts of propionic acid. Group IV organisms
produce only neurotoxin of type G.
[0029] It is known to obtain a tetanus toxin using specific media
substantially free of animal products. See e.g. U.S. Pat. No.
6,558,926. But notably, even the "animal product free" media
disclosed by this patent uses Bacto-peptone, a meat digest.
Significantly, production of tetanus toxin by Clostridium tetani
vs. production of a--botulinum toxin by a Clostridium botulinum
bacterium entails different growth, media and fermentation
parameters and considerations. See e.g. Johnson, E. A., et al.,
Clostridium botulinum and its neurotoxins: a metabolic and cellular
perspective, Toxicon 39 (2001), 1703-1722.
[0030] Production of Active Botulinum Neurotoxin
[0031] Botulinum toxin for use in a pharmaceutical composition can
be obtained 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.). Both the Schantz and the modified Schantz process
for obtaining a botulinum toxin make use of animal products.
[0032] A Clostridium botulinum neurotoxin (as pure toxin or as a
botulinum toxin complex) can also be obtained by aerobic
fermentation of a recombinant host cell which bears the appropriate
gene. See e.g. U.S. Pat. No. 5,919,665 entitled Vaccine for
clostridium botulinum neurotoxin, issued Jul. 6, 1999 to Williams
and U.S. patent application 20030215468 entitled Soluble
recombinant botulinum toxin proteins by Williams et al., published
Nov. 20, 2003.
[0033] Additionally, botulinum toxins (the 150 kilodalton molecule)
and botulinum toxin complexes (300 kDa to 900 kDa) can be obtained
from, for example, List Biological Laboratories, Inc., Campbell,
Calif.; the Centre for Applied Microbiology and Research, Porton
Down, U.K.; Wako (Osaka, Japan), as well as from Sigma Chemicals of
St Louis, Mo. Commercially available botulinum toxin containing
pharmaceutical compositions include Botox.RTM. (Botulinum toxin
type A purified neurotoxin complex with human serum albumin and
sodium chloride) available from Allergan, Inc., of Irvine, Calif.
in 100 unit vials as a lyophilized powder to be reconstituted with
0.9% sodium chloride before use), Dysport.RTM. (Clostridium
botulinum type A toxin hemagglutinin complex with human serum
albumin and lactose in the botulinum toxin pharmaceutical
composition), available from Ipsen Limited, Berkshire, U.K. as a
powder to be reconstituted with 0.9% sodium chloride before use),
and MyoBloc.TM. (an injectable solution comprising botulinum toxin
type B, human serum albumin, sodium succinate, and sodium chloride
at about pH 5.6, available from Solstice Neurosciences (formerly
available from Elan Corporation, Dublin, Ireland) of San Diego,
Calif.
[0034] A number of steps are required to make a Clostridial toxin
pharmaceutical composition suitable for administration to a human
or animal for a therapeutic, diagnostic, research or cosmetic
purpose. These steps can include obtaining a purified Clostridial
toxin and then compounding the purified Clostridial toxin. A first
step can be to culture a Clostridial bacteria, typically on agar
plates, in an environment conducive to bacterial growth, such as in
a warm anaerobic atmosphere. The culture step allows Clostridial
colonies with desirable morphology and other characteristics to be
obtained. In a second step selected cultured Clostridial colonies
can be fermented in a suitable medium. After a certain period of
fermentation the Clostridial bacteria typically lyse and release
Clostridial toxin into the medium. Thirdly, the culture medium can
be purified so as to obtain a bulk or raw toxin. Typically culture
medium purification to obtain bulk toxin is carried out using,
among other reagents, animal derived enzymes, such as DNase and
RNase, which are used to degrade and facilitate removal of nucleic
acids. The resulting bulk toxin can be a highly purified toxin with
a high specific activity. After stabilization in a suitable buffer,
the bulk toxin can be compounded with one or more excipients to
make a Clostridial toxin pharmaceutical composition suitable for
administration to a human. The Clostridial toxin pharmaceutical
composition can comprises a Clostridial toxin as an active
pharmaceutical ingredient. The pharmaceutical composition can also
include one or more excipients, buffers, carriers, stabilizers,
preservatives and/or bulking agents.
[0035] The Clostridium toxin fermentation step can result in a
culture solution which contains whole Clostridium bacteria, lysed
bacteria, culture media nutrients and fermentation byproducts.
Filtration of this culture solution so as to remove gross elements,
such as whole and lysed bacteria, provides a clarified culture. The
clarified culture solution comprises a Clostridial and various
impurities and can be processed so as to obtain a concentrated
Clostridial toxin, which is called bulk toxin.
[0036] Fermentation and purification processes for obtaining a bulk
Clostridial toxin using one or more animal derived products (such
as the milk digest casein, DNase and RNase) are known. An example
of such a known non-APF process for obtaining a botulinum toxin
complex is the Schantz and modified Schantz processes. The Schantz
and modified Schantz processes (from initial cell culture through
to fermentation and toxin purification) make use of a number of
products derived from animal sources such as for example animal
derived Bacto Cooked Meat medium in the culture vial, Columbia
Blood Agar plates for colony growth and selection, and casein in
the fermentation media. Additionally, the Schantz bulk toxin
purification process makes use of DNase and RNase from bovine
sources to hydrolyze nucleic acids present in the toxin containing
fermented culture medium. Concerns has been expressed regarding a
potential for a viral and transmissible spongiform encephalopathy
(TSE), such as a bovine spongiform encephalopathy (BSE),
contamination when animal products are used in a process for
obtaining an active pharmaceutical ingredient (API) and/or in a
process for making (compounding) a pharmaceutical composition using
such an API.
[0037] A fermentation process for obtaining a tetanus toxoid which
uses reduced amounts of animal derived products (referred to as
animal product free or "APF" fermentation processes. APF
encompasses animal protein free) is known. See e.g. U.S. Pat. No.
6,558,926 entitled Method for production of tetanus toxin using
media substantially free of animal products, issued to Demain et
al., May 6, 2003. An APF fermentation process for obtaining a
Clostridial toxin, has the potential advantage of reducing the (the
already very low) possibility of contamination of the ensuing bulk
toxin with viruses, prions or other undesirable elements which can
then accompany the active pharmaceutical ingredient Clostridial
toxin as it is compounded into a pharmaceutical composition for
administration to humans.
[0038] Column Chromatography
[0039] Column chromatography can be used to separate a particular
protein (such as a botulinum toxin) from a mixture of proteins,
nucleic acids, cell debris, etc in a process known as fractionation
or purification. The protein mixture is passed through a glass or
plastic column containing a solid, often porous matrix (referred to
as beads or as a resin). Different proteins and other compounds
pass through the matrix at different rates based on their specific
chemical characteristics and the way in which these characteristics
cause them to interact with the matrix.
[0040] To carry out column chromatography the protein mixture and
the matrix are immersed in a solvent. The sample (protein mixture
in the solvent) is applied to the top of the column, and the
solvent allowed to drain through the column. One the sample has
entered the matrix, solvent is added as needed to prevent the
matrix from drying out. The solvent is collected into separate
tubes (fractions) as it drains out of the bottom of the column.
Various components of the protein mixture travel through the column
at different rates based on differences in their chemical
characteristics, and are thus eventually fractionated into the
different tubes. The choice of matrix determines the type of
chemical characteristic on which the fractionation of the proteins
is based. There are three basic types of column chromatography. Ion
exchange chromatography accomplishes fractionation is based on
electrostatic charge. The column is packed with small beads
carrying either a positive or a negative charge. The extent to
which a given protein binds to the column matrix is a function of
the charge characteristics of the individual proteins. Since
proteins differ in their amino acid composition they differ in net
charge. Bound proteins are then selectively washed from the column
using a solvent (the eluant) containing a charged substance
(usually salt ions) that compete with the matrix beads in binding
the charged proteins. The proteins with the weakest charge
interactions will be washed from the column by the eluant first. As
the concentration of charged ions in the eluant is gradually
increased, more and more highly charged proteins will be washed
from the matrix. Bound proteins are thus fractionated on the basis
of the strength of their charge.
[0041] With gel filtration chromatography proteins are fractionated
on the basis of their size. The column is packed with tiny, porous
beads. Protein molecules small enough to enter the beads have a
longer path as they travel through the column matrix than do larger
molecules. Protein molecules large enough to be excluded from the
matrix thus emerge in the flow-through fraction, while smaller
proteins emerge in later fractions, based on their size relative to
the bead pores. Finally in affinity chromatography proteins are
separated based on their ability to bind to specific chemical
groups (ligand) attached to beads in the column matrix. The ligands
can be biologically specific for a target protein. For example, the
ligand may be substrate for a particular type of enzyme. Bound
proteins are eluted from the column.
[0042] It is known to use column chromatography to purify
(fractionate) a Clostridial toxin. See for example the following
publications: [0043] 1. Ozutsumi K., et al, Rapid, simplified
method for production and purification of tetanus toxin, App &
Environ Micro, Apr. 1985, p 939-943, vol 49, no. 4. (1985)
discloses use of high pressure liquid chromatography (HPLC) gel
filtration to purify tetanus toxin. [0044] 2. 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 discloses use of size exclusion chromatography
or ion exchange chromatograph to purify botulinum toxin type E.
Also disclosed is use of protamine sulfate instead of ribonuclease
(RNase). [0045] 3. Simpson L. L., et al., Isolation and
characterization of the botulinum neurotoxins Simpson L L; Schmidt
J J; Middlebrook J L, In: Harsman S, ed. Methods in Enzymology.
Vol. 165, Microbial Toxins: Tools in Enzymology San Diego, Calif.:
Academic Press; vol 165:pages 76-85 (1988) discloses purification
of botulinum neurotoxins using gravity flow chromatography, HPLC,
capture steps using an affinity resin, size exclusion
chromatography, and ion (anion and cation) exchange chromatography,
including use of two different ion exchange columns. Various
Sephadex, Sephacel, Trisacryl, S and Q columns are disclosed.
[0046] 4. 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) discloses
use of an amylose affinity column to purify botulinum neurotoxin
light chain fusion proteins. [0047] 5. Kannan K., et al., Methods
development for the biochemical assessment of Neurobloc (botulinum
toxin type B), Mov Disord 2000; 15(Suppl 2):20 (2000) discloses use
of size exclusion chromatography to assay a botulinum toxin type B.
[0048] 6. 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) discloses ion exchange chromatography to
purify a botulinum toxin type A. This reference discloses a
combination of precipitation and chromatography techniques. [0049]
7. 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) discloses use of ion
exchange and hydrophobic interaction columns to purify a
recombinant heavy chain fragment of a botulinum toxin type F.
[0050] 8. Published U.S. patent application 2003 0008367 A1 (Oguma)
discloses use of ion exchange and lactose columns to purify a
botulinum toxin.
[0051] The purification methods summarized above relate generally
to research or laboratory scale methods which are not scaleable
into industrial or commercial processes. It is well known that
chromatography techniques such as, for example, gel filtration and
gravity flow chromatography are not amenable for use as
large-scale, validatable, cGMP manufacturing processes. Alternately
or in addition, the purification method summarized above relate to
small scale purification of the neurotoxic component of a botulinum
toxin complex (i.e. the approximately 150 kDa neurotoxic molecule),
or a specific component of the neurotoxic component, as opposed to
purification of the entire 900 kDa botulinum toxin complex. As is
also well known, obtaining a biologically active, purified
botulinum toxin complex is considerably more complex and difficult,
than is purifying only a component of the complex. This is due, for
example, to the larger size, fragility, labile nature and
particular secondary, tertiary and quaternary molecule and complex
conformations required for obtaining a biologically active and
stable botulinum toxin complex.
[0052] Furthermore, existing processes, including commercial scale
processes, for obtaining a botulinum toxin suitable for compounding
into a botulinum toxin pharmaceutical composition typically include
a series of precipitation steps to separate the toxin complex from
impurities which accompany the botulinum toxin from the
fermentation process. Notably, precipitation techniques are widely
used in the biopharmaceutical industry to purification a botulinum
toxin. For example, cold alcohol fractionation (Cohn's method) or
precipitation is used to remove plasma proteins. Unfortunately,
precipitation techniques for purifying a botulinum toxin have the
drawbacks of low resolution, low productivity, difficulty to
operate, difficulty to control and/validate, difficulty to scale-up
or scale-down.
[0053] What is needed therefore is an APF process for purifying a
Clostridial toxin fermentation medium so as to obtain a bulk
Clostridial toxin without making use of animal derived products in
the purification process.
SUMMARY
[0054] Our invention provides various chromatographic APF systems
and processes for purifying a Clostridial toxin. The systems and
processes of our invention are scalable and cGMP compliant. The
Clostridial toxin is preferably a botulinum toxin, and most
preferably a botulinum toxin type A 900 kDa complex. The present
invention can be used as a commercial, industrial scale APF
purification process, to purify the Clostridial toxin (such as
botulinum toxin) obtained from a separate APF fermentation (i.e.
use of soy instead of casein in the fermentation medium) of a
Clostridial bacterium. The present invention therefore permits
replacement of the non-APF purification (i.e. use of DNase and
RNase) process, which is typically carried out after a non-APF
fermentation, to purify the botulinum toxin.
[0055] The present invention can be also have utility subsequent to
a Schantz fermentation of a Clostridial bacterium, to replace the
Schantz (non-APF) purification process, with the herein disclosed
APF toxin purification process. It is not preferred to practice the
present invention after a non-APF fermentation process, as opposed
to practicing the present invention after an APF fermentation
process, because the present invention has been optimized for use
subsequent to an APF fermentation process.
[0056] Thus, processes within the scope of the present invention
are preferably used in conjunction with (subsequent to) an APF
fermentation to thereby further reduce, and in certain embodiments
eliminate, use of animal derived products in the steps required to
obtain a bulk Clostridial toxin. Clearly practice of the present
invention subsequent to an APF fermentation process permits an
essentially completely APF methodology (fermentation and
purification) to be carried out.
[0057] An embodiment of the present invention provides a system and
process for obtaining high yield of highly purifying biologically
active Clostridial toxin. The present invention accomplishes this
through use of a free or substantially animal product free
chromatographic system and process to purify a clarified culture
obtained from the fermentation processes of a Clostridium
bacterium, such as a Clostridium botulinum bacterium.
DEFINITIONS
[0058] As used herein, the words or terms set forth below have the
following definitions.
[0059] "About" means that the item, parameter or term so qualified
encompasses a range of plus or minus ten percent above and below
the value of the stated item, parameter or term.
[0060] "Administration," or "to administer" means the step of
giving (i.e. administering) a pharmaceutical composition to a
subject. The pharmaceutical compositions disclosed herein are
"locally administered" by e.g. intramuscular (i.m.), intradermal,
subcutaneous administration, intrathecal administration,
intracranial intraperitoneal (i.p.) administration, topical
(transdermal) and implantation (i.e. of a slow-release device such
as polymeric implant or miniosmotic pump) routes of
administration.
[0061] "Animal product free" or "substantially animal product free"
encompasses, respectively, "animal protein free" or "substantially
animal protein free" and means the absence or substantial absence
of blood derived, blood pooled and other animal derived products or
compounds. "Animal" means a mammal (such as a human), bird,
reptile, fish, insect, spider or other animal species. "Animal"
excludes microorganisms, such as bacteria. Thus, an animal product
free medium or process or a substantially animal product free
medium or process within the scope of the present invention can
include a botulinum toxin or a Clostridial botulinum bacterium. For
example, an animal product free process or a substantially animal
product free process means a process which is either substantially
free or essentially free or entirely free of animal derived
proteins, such as immunoglobulins, meat digest, meat by products
and milk or dairy products or digests. Thus, an example of an
animal product free process is a process (such as a bacterial
culturing or bacterial fermentation process) which excludes meat
and dairy products or meat or dairy by products.
[0062] "Botulinum toxin" means a neurotoxin produced by Clostridium
botulinum, as well as modified, recombinant, hybrid and chimeric
botulinum toxins. A recombinant botulinum toxin can have the light
chain and/or the heavy chain thereof made recombinantly by a
non-Clostridial species. "Botulinum toxin," as used herein,
encompasses the botulinum toxin serotypes A, B, C, D, E, F and G.
"Botulinum toxin," as used herein, also encompasses both a
botulinum toxin complex (i.e. the 300, 600 and 900 kDa complexes)
as well as pure botulinum toxin (i.e. the about 150 kDa neurotoxic
molecule), all of which are useful in the practice of the present
invention. "Purified botulinum toxin" means a pure botulinum toxin
or a botulinum toxin complex that is isolated, or substantially
isolated, from other proteins and impurities which can accompany
the botulinum toxin as it is obtained from a culture or
fermentation process. Thus, a purified botulinum toxin can have at
least 90%, preferably more than 95%, and most preferably more than
99% of the non-botulinum toxin proteins and impurities removed. The
botulinum C.sub.2 and C.sub.3 cytotoxins, not being neurotoxins,
are excluded from the scope of the present invention.
[0063] "Clostridial neurotoxin" means a neurotoxin produced from,
or native to, a Clostridial bacterium, such as Clostridium
botulinum, Clostridium butyricum or Clostridium beratti, as well as
a Clostridial neurotoxin made recombinantly by a non-Clostridial
species.
[0064] "Entirely free (i.e. "consisting of" terminology) means that
within the detection range of the instrument or process being used,
the substance cannot be detected or its presence cannot be
confirmed.
[0065] "Essentially free" (or "consisting essentially of") means
that only trace amounts of the substance can be detected.
[0066] "Modified botulinum toxin" means a botulinum toxin that has
had at least one of its amino acids deleted, modified, or replaced,
as compared to a native botulinum toxin. Additionally, the modified
botulinum toxin can be a recombinantly produced neurotoxin, or a
derivative or fragment of a recombinantly made neurotoxin. A
modified botulinum toxin retains at least one biological activity
of the native botulinum toxin, such as, the ability to bind to a
botulinum toxin receptor, or the ability to inhibit
neurotransmitter release from a neuron. One example of a modified
botulinum toxin is a botulinum toxin that has a light chain from
one botulinum toxin serotype (such as serotype A), and a heavy
chain from a different botulinum toxin serotype (such as serotype
B). Another example of a modified botulinum toxin is a botulinum
toxin coupled to a neurotransmitter, such as substance P.
[0067] "Patient" means a human or non-human subject receiving
medical or veterinary care. Accordingly, as disclosed herein, the
compositions may be used in treating any animal, such as
mammals.
[0068] "Pharmaceutical composition" means a formulation in which an
active ingredient can be a botulinum toxin. The word "formulation"
means that there is at least one additional ingredient (such as an
albumin [such as a human serum albumin or a recombinant human
albumin] and/or sodium chloride) in the pharmaceutical composition
besides a neurotoxin active ingredient. A pharmaceutical
composition is therefore a formulation which is suitable for
diagnostic, therapeutic or cosmetic administration (i.e. by
intramuscular or subcutaneous injection or by insertion of a depot
or implant) to a subject, such as a human patient. The
pharmaceutical composition can be: in a lyophilized or vacuum dried
condition; a solution formed after reconstitution of the
lyophilized or vacuum dried pharmaceutical composition with saline
or water, or; as a solution which does not require reconstitution.
The active ingredient can be one of the botulinum toxin serotypes
A, B, C.sub.1, D, E, F or G or a botulinum toxin, all of which can
be made natively by Clostridial bacteria. As stated, a
pharmaceutical composition can be liquid or solid, for example
vacuum-dried. The constituent ingredients of a pharmaceutical
composition can be included in a single composition (that is all
the constituent ingredients, except for any required reconstitution
fluid, are present at the time of initial compounding of the
pharmaceutical composition) or as a two-component system, for
example a vacuum-dried composition reconstituted with a diluent
such as saline which diluent contains an ingredient not present in
the initial compounding of the pharmaceutical composition. A
two-component system provides the benefit of allowing incorporation
of ingredients which are not sufficiently compatible for long-term
shelf storage with the first component of the two component system.
For example, the reconstitution vehicle or diluent may include a
preservative which provides sufficient protection against microbial
growth for the use period, for example one-week of refrigerated
storage, but is not present during the two-year freezer storage
period during which time it might degrade the toxin. Other
ingredients, which may not be compatible with a Clostridial toxin
or other ingredients for long periods of time, may be incorporated
in this manner; that is, added in a second vehicle (i.e. in the
reconstitution fluid) at the approximate time of use. Methods for
formulating a botulinum toxin active ingredient pharmaceutical
composition are disclosed in U.S. patent publication 2003 0118598
A1.
[0069] "Substantially free" means present at a level of less than
one percent by weight of a culture medium, fermentation medium,
pharmaceutical composition or other material in which the weight
percent of a substance (such as an animal product, animal protein
or animal derived product or protein is assessed.
[0070] "Therapeutic formulation" means a formulation can be used to
treat and thereby alleviate a disorder or a disease, such as a
disorder or a disease characterized by hyperactivity (i.e.
spasticity) of a peripheral muscle.
[0071] The following abbreviations are used herein: [0072] 3:1:1
culture a botulinum toxin culture/fermentation medium containing 3%
HySoy, 1% HyYeast, and 1% glucose. HySoy (Quest product no.
5.times.59022) is a source of peptides made by enzymatic hydrolysis
of soy. HyYeast (HyYest, Quest product no. 5Z10102 or 5Z10313 is a
baker's yeast extract. [0073] 5:1:1 culture a botulinum toxin
culture/fermentation medium containing 5% HySoy, 1% HyYeast, and 1%
glucose. [0074] API active pharmaceutical ingredient [0075] APF
animal product free [0076] BCA bicinchoninic acid [0077] CV column
volume [0078] DF diafiltration [0079] ELISA enzyme linked
immunosorbent assay. "Hc" in "Hc-ELISA means a botulinum toxin
heavy chain. [0080] MLD50 the amount of a botulinum toxin which is
a lethal dose to 50% of 18-23 gram Swiss-Weber mice injected
intraperitoneally [0081] SDS-PAGE sodium
dodecylsulfate-polyacrylamide gel electrophoresis [0082] SEC-HPLC
size exclusion high performance liquid chromatography [0083] UF
ultrafiltration [0084] UV ultraviolet
[0085] Our invention includes a process for purifying a Clostridium
toxin. The process can have the four steps of obtaining a sample of
a botulinum toxin fermentation culture; contacting a first
chromatography column resin with the culture sample so as to permit
capture of a botulinum toxin by the first column; eluting the
botulinum toxin from the first column, and; loading a second column
chromatography column resin with the eluent from the first
chromatography column, thereby obtaining a purified botulinum
toxin. By "botulinum toxin fermentation culture" it is meant a
fermentation medium in which a Clostridium botulinum bacterium has
been fermented so that the bacterium has released botulinum toxin
into the medium. The sample of a botulinum toxin fermentation
culture (medium) is preferably a sample of a clarified culture of
the fermentation medium.
[0086] The first chromatography column and the second
chromatography column can be different columns, and the two
different columns can act to purify a botulinum toxin through
different purification mechanisms. For example, the first
chromatography column can be a hydrophobic interaction column and
the second chromatography column can be an ion exchange column.
[0087] A process for purifying a Clostridium toxin within the scope
of our invention can also have the step after the contacting step
and before the eluting step, of washing impurities off the first
column. Additionally, a process for purifying a Clostridium toxin
within the scope of our invention can also have the step after the
loading step, the step of washing impurities off the second column.
Furthermore, a process for purifying a Clostridium toxin within the
scope of our invention can also have, after the step of washing
impurities off the second column, the step of eluting the botulinum
toxin from the second column.
[0088] Preferably, a process for purifying a Clostridium toxin
within the scope of our invention is a substantially animal product
free (APF) process, such as a substantially animal protein free
process, and more preferably it is an essentially APF process for
purifying a clostridial toxin, such as a botulinum toxin complex.
Most preferably, a process for purifying a Clostridium toxin within
the scope of our invention is an animal product free process.
[0089] The botulinum toxin fermentation culture used in a process
for purifying a Clostridium toxin within the scope of our invention
preferably results from an APF process, more preferably results
from a substantially APF process, and most preferably results from
an essentially APF process.
[0090] Significantly, a process for purifying a Clostridium toxin
within the scope of our invention can preferably provide a yield of
more than about 20 mg of high quality purified botulinum toxin
complex per batch for each 10 liters of a botulinum toxin
fermentation culture (APF clarified culture), more preferably more
than about 30 mg of high quality purified botulinum toxin complex
per 10 liters of botulinum toxin clarified culture, and most
preferably more than about 40 mg of high quality purified botulinum
toxin complex per 10 liters clarified culture. A particularly
preferred embodiment of the present invention can provide 50 mg or
more of high quality purified botulinum toxin complex per 10 liters
of botulinum toxin clarified culture. High quality means with at
least the potency ("Specific Potency") and purity ("Nucleic Acids")
characteristics set forth in Table 1.
[0091] A purified botulinum toxin complex obtained by practice of a
process for purifying a Clostridium toxin within the scope of our
invention can have the following characteristics: an appearance as
an white to off-white suspension; a concentration of 2.0-3.6 mg of
botulinum toxin complex per ml of eluent; the ratio of absorbance
at 260 nm to absorbance at 278 nm (A260/A278) is less than or equal
to 0.6; a specific potency in MLD50 unit/mg of between
2.4.times.10.sup.7 to 5.9.times.10.sup.7 MLD50 units per mg of the
purified botulinum toxin; an immunological identity to botulinum
neurotoxin type A complex; an SDS-PAGE characteristic that conforms
to standard; an SEC-HPLC characteristic of 900 kDa toxin complex of
>95% of the total peak, and; the process used to obtain such a
purified botulinum toxin complex is robust, scalable, validatable,
and/or cGMP compliant.
[0092] An APF process for purifying a botulinum toxin complex
within the scope of our invention can have the steps of:
[0093] (a) obtaining a sample of a botulinum toxin fermentation
culture, wherein the botulinum toxin fermentation culture results
from a substantially APF process.
[0094] (b) contacting a hydrophobic interaction chromatography
column resin with the culture sample so as to permit capture of a
botulinum toxin by the first column;
[0095] (c) washing impurities off the hydrophobic interaction
chromatography column;
[0096] (d) eluting the botulinum toxin from the hydrophobic
interaction column (the eluting step can be followed by the step of
diluting the eluent from the hydrophobic interaction chromatography
column for a subsequent ion exchange chromatography);
[0097] (e) loading an ion exchange column chromatography column
resin with the eluent (such as the diluted eluent from the
hydrophobic interaction chromatography column) from the hydrophobic
interaction chromatography column;
[0098] (f) washing impurities off the ion exchange chromatography
column, and;
[0099] (g) eluting the botulinum toxin from the ion change column,
thereby obtaining a purified botulinum toxin through a process for
purifying a botulinum toxin which is a substantially APF
purification process.
[0100] The APF process set forth in the paragraph above can further
comprise, after the step of obtaining a sample of a botulinum toxin
fermentation culture and before the step of contacting a
hydrophobic interaction chromatography column resin with the
culture sample, the additional step of conditioning the clarified
culture for hydrophobic interaction chromatography. Additionally,
the APF process set forth in the paragraph above can further
comprise, after the step of eluting the botulinum toxin from the
hydrophobic interaction column and before the step of loading an
ion exchange column chromatography column resin with the eluent
from the hydrophobic interaction chromatography column, the step of
conditioning the eluent from hydrophobic interaction column for ion
exchange chromatography.
[0101] A detailed embodiment of an APF process for purifying a
botulinum toxin, the process can comprise the steps of:
[0102] (a) obtaining a sample of a botulinum toxin fermentation
culture, wherein the botulinum toxin fermentation culture results
from a substantially APF process.
[0103] (b) conditioning the clarified culture for hydrophobic
interaction chromatography;
[0104] (c) contacting a hydrophobic interaction chromatography
column resin with the culture sample so as to permit capture of a
botulinum toxin by the first column;
[0105] (d) washing impurities off the hydrophobic interaction
chromatography column;
[0106] (e) eluting the botulinum toxin from the hydrophobic
interaction column;
[0107] (f) conditioning the eluent from hydrophobic interaction
column for ion exchange chromatography;
[0108] (g) loading an ion exchange column chromatography column
resin with the conditioned eluent from the hydrophobic interaction
chromatography column;
[0109] (h) washing impurities off the ion exchange chromatography
column, and;
[0110] (i) eluting the botulinum toxin from the ion change column,
thereby obtaining a purified botulinum toxin through a process for
purifying a botulinum toxin which is a substantially APF
purification process.
[0111] Also within the scope of our invention is a system for
purifying a Clostridium toxin, such as a botulinum toxin type A
complex. Such a system can comprise: a first chromatography column
resin for capturing a botulinum toxin from a fermentation culture;
an elution buffer for eluting the botulinum toxin from the first
column; a second column chromatography column resin for capturing a
botulinum toxin from an eluent from the first chromatography
column, and; a second elution buffer for eluting the botulinum
toxin from the second chromatography column.
[0112] Our invention also encompasses a process for purifying a
botulinum toxin, the process comprising the steps of: (a) obtaining
a fermentation medium which comprises a botulinum toxin; (b)
filtering the fermentation medium; (c) precipitating botulinum
toxin from the filtered fermentation medium to thereby obtain a
precipitate; (d) extracting botulinum toxin from the precipitate;
(e) contacting a first chromatography column with the extracted
botulinum toxin; (f) eluting the botulinum toxin from the first
chromatography column; (g) contacting a second column
chromatography column with the botulinum toxin containing eluent
from the first chromatography column; (h) eluting the botulinum
toxin from the second chromatography column; (i) contacting a third
column chromatography column with the botulinum toxin containing
eluent from the second chromatography column, and; (j) eluting the
botulinum toxin from the third chromatography column, thereby
obtaining a purified botulinum toxin. In this process for purifying
a botulinum toxin the first chromatography column, the second
chromatography column, and the third chromatography column can be
different chromatography columns and the process can be a process
which is a substantially animal product and/or animal protein free
(APF) process, an essentially APF process or an APF process.
[0113] Additionally, in the process for purifying a botulinum toxin
of claim set forth in the paragraph above the botulinum toxin
fermentation medium can result from a substantially APF
fermentation process, an essentially APF fermentation process or
from an APF fermentation process. Significantly, this process for
purifying a botulinum toxin can result in a purified botulinum
toxin obtained which has a potency of up to about 100 million units
per milligram of the purified botulinum toxin, as little as about
30 ng of residual nucleic acids per mg of the purified botulinum
toxin and/or a ratio of absorbance at 260 nm to it's absorbance at
278 nm (A260/A278) as low as about 0.50.
[0114] A detailed embodiment of our APF process for purifying a
botulinum toxin, the process can comprise the steps of: (a)
obtaining an APF fermentation medium which comprises a botulinum
toxin; (b) filtering the APF fermentation medium; (c) precipitating
botulinum toxin from the filtered APF fermentation medium to
thereby obtain a precipitate; (d) extracting botulinum toxin from
the precipitate; (e) contacting a first chromatography column with
the extracted botulinum toxin; (f) eluting the botulinum toxin from
the first chromatography column; (g) contacting a second column
chromatography column with the botulinum toxin containing eluent
from the first chromatography column; (h) eluting the botulinum
toxin from the second chromatography column; (i) contacting a third
column chromatography column with the botulinum toxin containing
eluent from the second chromatography column, and; (j) eluting the
botulinum toxin from the third chromatography column, thereby
obtaining a purified botulinum toxin, wherein the first
chromatography column and again the second chromatography column,
and the third chromatography column can be different chromatography
columns.
[0115] Our invention also includes a APF system for purifying a
botulinum toxin, the APF system comprising: (a) a first
chromatography column for capturing a botulinum toxin obtained from
an extract obtained by (i) filtering an APF fermentation medium,
(ii) precipitating botulinum toxin from the filtered APF
fermentation medium, and (iii) extracting botulinum toxin from the
precipitate; (b) an elution buffer for eluting the botulinum toxin
from the first chromatography column; (c) a second column
chromatography column resin for capturing a--botulinum toxin from
an eluent from the first chromatography column; (d) a second
elution buffer for eluting the botulinum toxin from the second
chromatography column; (e) a third chromatography column for
capturing a botulinum toxin from the eluent from the second
chromatography column, and; (f) a third elution buffer for eluting
the botulinum toxin from the third chromatography column;
DRAWINGS
[0116] Aspects of the invention are explained or illustrated by the
following drawings.
[0117] FIG. 1 entitled N-Source (i.e. HySoy plus YE) % vs. Potency
and pH" is a graph showing botulinum toxin activity as determined:
(1) on the left side Y axis mouse lethal dose 50 (MLD 50) (blue
bars), and; (2) on the left side Y axis SNAP 25 activity (red
bars), of various APF media at the elapsed fermentation times shown
at the top of the bars, for APF medium pH as shown on the right
side Y axis the pH, for APF media with the wt % amount of
hydrolyzed soy concentrate and yeast extract concentrate as shown
by the X axis. All FIG. 1 media also contained 1% by wt
glucose.
[0118] FIG. 2 is a summary flow chart comparing a non-APF process
for obtaining a botulinum toxin (the top half of FIG. 1) with an
APF process, within the scope of the present invention, for
obtaining a botulinum toxin (the bottom half of FIG. 2), through
the cell bank creation, culture and fermentation steps. FIG. 2
omits the harvest and purification steps.
[0119] FIG. 3 is a chromatograph obtained from hydrophobic
interaction chromatography of an APF clarified culture (a 3.1.1
culture) on a Butyl Sepharose Fast Flow column. The X axis in FIG.
3 represents the volume in ml of liquid (effluent) which has passed
through the column. The Y axis represents absorbance at 280 nm in
mAU.
[0120] FIG. 4 is a chromatograph obtained from ionic exchange
chromatography of the eluent from the FIG. 3 Butyl column on an SP
Sepharose high performance column. The axes in FIG. 4 are the same
as they are for FIG. 3.
[0121] FIG. 5A is an image of reduced SDS-PAGE of various fractions
obtained from operation of the Butyl column of FIG. 3. The left
hand side of FIG. 5A is marked vertically with descending molecular
weights in thousands of Daltons (kDa). The numbers 1 to 8 along the
bottom border of FIG. 5A represents the lanes in which fractions
were loaded.
[0122] FIG. 5B is an image of reduced SDS-PAGE of various fractions
obtained from operation of the SP column of FIG. 4. The left and
bottom sides of FIG. 5B are marked as they are in FIG. 5A.
[0123] FIG. 6 is an image of reduced SDS-PAGE of various fractions
obtained in post column steps (see FIG. 7), namely fractions from
the UF/DF step, the sterile filtration step, and from the ammonium
sulfate precipitation step. The left and bottom sides of FIG. 6 are
marked as they are in FIG. 5A.
[0124] FIG. 7 is a flow chart of a APF chromatographic botulinum
toxin purification process within the scope of the present
invention.
[0125] FIG. 8 is a graph comparing the effect of a soy protein
concentration on a botulinum toxin type A complex production in an
APF fermentation process, where the fermentation medium contained 1
wt % glucose and 1 wt % of a yeast extract. In FIG. 8 the X axis
represents the weight percent concentration in the fermentation
medium of a particular hydrolyzed soy protein (HySoy), the left
side Y axis represents potency of the final purified botulinum
toxin complex and the right side Y axis represents the percent of
cell lysis completed, as determined by the equation:
Cell Lysis ( % ) = OD 600 max - OD 600 endpoint OD 600 max .times.
100 ##EQU00001##
where OD.sub.600 max corresponds to the optical density measured at
600 nm at the time of maximum growth, and OD.sub.600 endpoint is at
the time of fermentation harvest.
[0126] FIG. 9 illustrates in summary flow chart fashion significant
distinctions between the Example 6 non-APF purification process, a
preferred embodiment of the Examples 12-15 purification process and
a preferred embodiment of the Example 16 botulinum toxin clarified
culture purification process.
[0127] FIG. 10 is an image of reduced SDS-PAGE of several
supernatants obtained from the Day 1 UF/DF process set forth in
Example 16, showing as well several molecular weight standards and
toxins. The numbers 1 to 9 along the bottom border of FIG. 10
represent nine lanes of materials.
DESCRIPTION
[0128] The present invention is based upon the discovery that a
Clostridial toxin can be purified by use of an animal product fee
(APF) system and process. The present invention encompasses a
animal product free system and process for purifying a Clostridium
botulinum neurotoxin. The Clostridium botulinum neurotoxin can be a
botulinum toxin type A complex, such as a 300 kD, 500 kD or 900 kD
(approximate molecular weights) complex or mixtures thereof. The
Clostridium botulinum neurotoxin can be any one of the serotypes A,
B, C, D, E, F or G or mixtures thereof. Additionally, the system
and process can be practiced in conjunction with a recombinant,
hybrid, chimeric or modified botulinum toxin (light chain, heavy
chain, or both chains together).
[0129] Significantly, the system and process disclosed herein is
scalable, meaning that it can be used to purify the quantities of
botulinum toxin obtained from an industrial or commercial process,
as use for pharmaceutical production. Further, the system and
process is also cGMP (current good manufacturing practices)
compliant, as required by the U.S. CFR (United States code of
federal regulations), meaning that it can comply with regulatory
requirements.
[0130] Through experimentation there was developed APF systems and
processes to purify a Clostridial toxin, such as a Clostridium
botulinum type A (Hall strain) neurotoxin complex. The Clostridial
toxin is purified from the fermentation medium resulting from
either a Schantz (non-APF) fermentation process or from an APF
fermentation process. Schantz processes use animal derived
products. Significantly, while an APF fermentation process can
reduce or eliminate animal derived products (such as casein and
meat broth) as nutrients from the media used to culture and ferment
Clostridial bacteria, APF fermentation processes are typically
followed by one or more purification steps which make use of animal
derived products, such as the enzymes DNase and RNase. Purification
of the fermentation medium is required to obtain bulk Clostridial
toxin. Bulk Clostridial toxin (pure toxin or toxin complex) can be
used for compounding a Clostridial toxin pharmaceutical
composition.
[0131] Preferably, the present invention is practiced in
conjunction with an APF fermentation process. Practicing the
present invention in conjunction with an APF fermentation process
provides a combined APF fermentation process and an APF
purification process. Additionally, systems and method of the
present invention are optimized for operation upon an APF
fermentation medium, as opposed to a casein or other animal protein
based fermentation medium. Practicing the presently invention upon
a non-APF fermentation can result in a lower yield and/or a lower
potency of the purified botulinum toxin obtained.
[0132] Thus, although both the Schantz and APF botulinum toxin
purification processes use animal derived products such as
benzamidine to stabilize the botulinum toxin and DNase and RNase to
remove nucleic acids present with the botulinum toxin in the
fermentation medium (see e.g. Examples 6 and 7), our invention
permits a botulinum toxin can be purified without using such animal
derived products.
[0133] The present invention encompasses systems and processes for
purifying a Clostridial toxin, such as a botulinum toxin complex.
Typically a particular system within the scope of the present
invention is operated in conjunction with a particular process
within the scope of the present invention. A system within the
scope of the present invention can comprise a plurality (preferably
as a consecutive series) of chromatography steps. A process within
the scope of the present invention can comprise passing a
Clostridial toxin fermentation medium through the plurality of
chromatography columns to thereby obtain a highly purified and
highly potent Clostridial toxin. Such a purified Clostridial toxin
is suitable for compounding a Clostridial toxin pharmaceutical
composition. Important parameters of systems and processes within
the scope of the present invention include the particular columns,
buffers and operating (column running) conditions used.
[0134] A first broad step in a particular embodiment of the
invention can be to load a fermentation medium clarified culture
onto a hydrophobic interaction column (such as a Butyl Sepharose
Fast Flow ["FF"] column). This first column captures the
Clostridial toxin (such as a botulinum toxin complex) and allows
impurities to flow through the column. It was found that a
hydrophobic interaction column provided an efficient capture of a
botulinum toxin complex (a large protein with a particular tertiary
and quaternary structure) from fermentation medium with retention
of the biological activity of the botulinum toxin complex, while
also separating (flow through) of many impurities present with the
botulinum toxin in the fermentation medium. A suitable buffer is
used to elute the captured (bound) Clostridial toxin from the
hydrophobic interaction column.
[0135] In a second broad step in a particular embodiment of the
present invention, the eluent from the first column is loaded onto
a second column to further purify the Clostridial toxin. It was
found that preferably, if the second column provides a different
mechanism for separation of Clostridial toxin from impurities, then
a second column chromatography step can provide a further efficient
purification step. Thus, preferably, the second chromatography step
entails use of a different column, such as a SP Sepharose high
performance ["HP"] column.
[0136] In post chromatography (column) steps eluent from the second
column can then be further processed to obtain highly purified bulk
botulinum toxin complex. These additional processing steps can
include buffer exchange by ultrafiltration and diafiltration,
sterile filtration and preparation of an ammonium sulphate
suspension of the purified botulinum toxin complex.
[0137] Our invention encompasses a scalable and cGMP compliant
system and process for purifying a botulinum toxin, which can
result in obtaining a bulk botulinum toxin with the characteristics
set forth in Table 1.
TABLE-US-00002 TABLE 1 Purified Botulinum Neurotoxin
Characteristics Appearance White to off-white suspension
Concentration 2.0-3.6 mg/ml Nucleic Acids (A260/A278) Not more than
0.6 Specific Potency (MLD50 unit/mg) 2.4-5.9 .times. 10.sup.7
Immunological Identity Pass SDS-PAGE Conformed to standard SEC-HPLC
900 kDa toxin complex >95% total peak
[0138] A commercially available botulinum toxin containing
pharmaceutical composition is sold under the trademark BOTOX.RTM.
(available from Allergan, Inc., of Irvine, Calif.). BOTOX.RTM. has
the characteristics set forth in Table 1 above. BOTOX.RTM. consists
of a purified botulinum toxin type A complex, human serum albumin,
and sodium chloride packaged in sterile, vacuum-dried form. The
botulinum toxin type A is made from a culture of the Hall strain of
Clostridium botulinum grown in a medium containing N-Z amine casein
and yeast extract (i.e. non-APF process). The botulinum toxin type
A complex is purified from the culture solution by a series of
precipitation (including acid precipitation) steps to a crystalline
complex consisting of the active high molecular weight toxin
protein and an associated hemagglutinin protein. The crystalline
complex is re-dissolved in a solution containing saline and albumin
and sterile filtered (0.2 microns) prior to vacuum-drying.
BOTOX.RTM. can be reconstituted with sterile, non-preserved saline
prior to intramuscular injection. Each vial of BOTOX.RTM. contains
about 100 units (U) of Clostridium botulinum toxin type A complex,
0.5 milligrams of human serum albumin and 0.9 milligrams of sodium
chloride in a sterile, vacuum-dried form without a
preservative.
[0139] To reconstitute vacuum-dried BOTOX.RTM. sterile normal
saline without a preservative (0.9% Sodium Chloride injection) is
used by drawing up the proper amount of diluent in the appropriate
size syringe. Since BOTOX.RTM. is denatured by bubbling or similar
violent agitation, the diluent is gently injected into the vial. It
has been reported that BOTOX.RTM. has been administered thirty or
more days after reconstitution with little loss of potency. During
this time period, reconstituted BOTOX.RTM. is stored in a
refrigerator (2.degree. to 8.degree. C.). Reconstituted BOTOX.RTM.
is clear, colorless and free of particulate matter. The
vacuum-dried product is stored in a freezer at or below -5.degree.
C.
[0140] The present invention is based upon the discovery of media
and processes which are free or substantially free of an animal
product or an animal byproduct useful for culture and fermentation
of an organism (such as a Clostridium botulinum bacterium) capable
of producing biologically active botulinum toxin. The botulinum
toxin obtained can be used for making botulinum toxin active
ingredient pharmaceutical compositions. Thus, growth media are
disclosed herein which have significantly reduced levels of meat or
dairy by-products and preferred media embodiments are substantially
free of such animal products.
[0141] The present invention encompasses the surprising finding
that animal-based products are not required in media for growth of
Clostridium botulinum, and particularly that vegetable-based
products can replace animal-based products typically employed in
such media for the growth of Clostridium botulinum.
[0142] Media that are in current use for growth and fermentation of
bacteria usually comprise one or more animal derived ingredients,
such as cooked meat. In accordance with the present invention,
preferred media for growth of Clostridium botulinum contain animal
derived ingredients which comprise no more than about five to about
ten percent of the total weight of the media. More preferably,
media within the scope of the invention comprise no more than about
one to less than about five percent of the total weight of the
media of animal derived products. Most preferably, all media and
cultures used for the growth of Clostridium botulinum for the
production of botulinum toxin are completely free of animal derived
products. These media include but are not limited to media for
small and large scale fermentation of Clostridium botulinum, media
for growth of cultures of Clostridium botulinum used to inoculate
the seed (first) media and fermentation (second) media, as well as
and media used for long-term storage of cultures of Clostridium
botulinum (e.g. stock cultures).
[0143] In certain preferred embodiments of the invention, the media
for the growth of Clostridium botulinum and production of botulinum
toxin can comprise soy based products to replace animal derived
products. Alternately, instead of a soy based product there can be
used debittered seed of Lupinus campestris. It is known the protein
content of L. campestris seed is very similar to that of soybean.
Preferably, these media include soybean or of L. campestris derived
products that are hydrolyzed and that are soluble in water.
However, insoluble soy or of L. campestris products can also be
used in the present invention to replace animal products. Common
animal derived products which can be substituted by soy or of L.
campestris products include beef heart infusion (BHI), animal
derived peptone products, such as Bacto-peptone, hydrolyzed
caseins, and dairy by-products such as animal milk.
[0144] Preferably media containing soy-based or of L. campestris
based products for the growth of Clostridium botulinum are similar
to commonly used growth media containing animal derived products
except that substantially all animal-derived products are replaced
with vegetable-derived products. For example, soy based
fermentation media 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,
KH.sub.2PO.sub.4, divalent cations such as iron and magnesium, iron
powder, and amino acids such as L-cysteine and L-tyrosine. Media
used to grow cultures of Clostridium botulinum for inoculation
(i.e. the seed or first medium) of the fermentation (second) media
preferably contain at least a soy based product, a source of salt
such as NaCl, and a carbon source such as glucose.
[0145] The present invention provides a method for the growth of
Clostridium botulinum that maximizes the production of a botulinum
toxin using media that are substantially free of animal-derived
products. Growth of Clostridium botulinum for production of
botulinum toxin can take place by fermentation in media containing
soy by-products that replace ingredients derived from animal
by-products. The inoculant for the fermentation medium can be
derived from a smaller scaled growth medium (a seed medium).
Depending on the size and volume of the fermentation step, the
number of successive growths in seed media to increase the biomass
of the culture can vary. To grow a suitable amount of Clostridium
botulinum for inoculating the fermentation medium, one step or
multiple steps involving growth in a seed medium can be performed.
For a method of growing Clostridium botulinum that is free of
animal derived products, it is preferable that growth of
Clostridium botulinum originates from a culture stored in non
animal derived media. The stored culture, preferably lyophilized,
is produced by growth in media containing proteins derived from soy
and lacking animal by-products. Growth of Clostridium botulinum in
a fermentation medium can take place by inoculation directly from a
stored, lyophilized culture.
[0146] In a preferred embodiment of the present invention, growth
of Clostridium botulinum proceeds in two phases-seed growth and
fermentation. Both of these phases are carried out in anaerobic
environments. The seed growth phase is generally used to "scale-up"
the quantity of the microorganism from a stored culture. The
purpose of the seed growth phase) is to increase the quantity of
the microorganism available for fermentation. In addition, the seed
growth phase allows relatively dormant microbes in stored cultures
to rejuvenate and grow into actively growing cultures. Furthermore,
the volume and quantity of viable microorganisms used to inoculate
the fermentation culture can be controlled more accurately from an
actively growing culture than from a stored culture. Thus, growth
of a seed culture for inoculation of the fermentation medium is
preferred. In addition, any number of consecutive steps involving
growth in seed media to scale-up the quantity of Clostridium
botulinum for inoculation of the fermentation medium can be used.
It is noted that growth of Clostridium botulinum in the
fermentation phase can proceed directly from the stored culture by
direct inoculation.
[0147] In the fermentation phase, a portion of a seed medium or all
of a seed medium containing Clostridium botulinum from the seed
growth is used to inoculate a fermentation medium. Preferably,
approximately 2-4% of a seed medium having Clostridium botulinum
from the seed growth phase is used to inoculate the fermentation
medium. Fermentation is used to produce the maximum amount of
microbe in a large-scale anaerobic environment (Ljungdahl et al.,
Manual of industrial microbiology and biotechnology (1986), edited
by Demain et al, American Society for Microbiology, Washington,
D.C. page. 84).
[0148] A botulinum toxin can be isolated and purified using methods
of protein purification well known to those of ordinary skill in
the protein purification art. See e.g. Coligan et al. Current
Protocols in Protein Science, Wiley & Sons; Ozutsumi et al.
Appl. Environ. Microbiol. 49; 939-943:1985.
[0149] For production of botulinum toxin, cultures of Clostridium
botulinum can be grown in a seed medium for inoculation of the
fermentation medium. The number of successive steps involving
growth in a seed medium can vary depending on the scale of the
production of botulinum toxin in the fermentation phase. However,
as previously discussed, growth in the fermentation phase may
proceed directly from inoculation from a stored culture.
Animal-based seed media generally are comprised of BHI,
bacto-peptone, NaCl, and glucose for growth of Clostridium
botulinum. As previously discussed, alternative seed media may be
prepared in accordance with the present invention in which
animal-based components are substituted with non-animal-based
components. For example but without limitation, soy-based products
can substitute for BHI and bacto-peptone in the seed medium for
growth of Clostridium botulinum and production of botulinum toxin.
Preferably, the soy-based product is soluble in water and comprises
hydrolyzed soy, although cultures of Clostridium botulinum can grow
in media containing insoluble soy. However, levels of growth and
subsequent toxin production are greater in media derived from
soluble soy products.
[0150] Any source of soy-based products may be used in accordance
with the present invention. Preferably, the soy is hydrolyzed soy
and the hydrolyzation has been carried out using non-animal
enzymes. Sources of hydrolyzed soy are available from a variety of
commercial vendors. These 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). Most
preferably, the source of hydrolyzed soy is Hy-Soy or SE50MK. Other
potential sources of hydrolyzed soy are known.
[0151] Concentrations of Hy-Soy in the seed medium in accordance
with the present invention range between 25-200 g/L. Preferably,
the concentration of Hy-Soy in the seed medium ranges between
50-150 g/L. Most preferably the concentration of Hy-Soy in the seed
medium is approximately 100 g/L. In addition, the concentration of
NaCl ranges between 0.1-2.0 g/L. Preferably the concentration of
NaCl ranges between 0.2-1.0 g/L. Most preferably, the concentration
of NaCl in the seed medium is approximately 0.5 g/L. The
concentration of glucose ranges between 0.1 g/L and 5.0 g/L.
Preferably, the concentration of glucose ranges between 0.5-2.0
g/L. Most preferably, the concentration of glucose in the seed
medium is approximately 1.0 g/L. It is also preferred but not
necessary for the present invention that the glucose is sterilized
by autoclaving together with the other components of the seed
medium. The pH level of the seed medium prior to growth can be
7.5-8.5. For example, the pH of the seed medium prior to growth of
Clostridium botulinum can be approximately 8.1.
[0152] Growth of Clostridium botulinum in the seed medium can
proceed in one or more stages. Preferably, growth in the seed
medium proceeds in two stages. In stage one, a culture of
Clostridium botulinum is suspended in a quantity of seed medium and
incubated at 34.+-.1.degree. C. for 24-48 hours in an anaerobic
environment. Preferably, growth in stage one proceeds for
approximately 48 hours. In stage two, a portion or all of the stage
one medium containing Clostridium botulinum is used to inoculate a
stage two seed medium for further growth. After inoculation, the
stage two medium is incubated at 34.+-.1.degree. C. for
approximately 1-4 days also in an anaerobic environment.
Preferably, growth in the stage two seed medium proceeds for
approximately 3 days. It is also preferable that growth in seed
media in any stage does not result in cell lysis before inoculation
of fermentation media with the final growth in seed medium.
[0153] Standard fermentation media containing animal by-products
for the growth of Clostridium botulinum can be based on a recipe of
Mueller and Miller (MM; J. Bacteriol. 67:271, 1954). The
ingredients in MM media containing animal by-products include BHI
and NZ-CaseTT. NZ-CaseTT is a commercially available source of
peptides and amino acids which are derived from the enzymatic
digestion of caseins, a group of proteins found in animal milk. The
present invention demonstrates that non-animal based products may
be substituted for BHI and NZ-CaseTT in fermentation media. For
example but without limitation, --soy-based products can replace
the animal-based components of MM media used for fermentation of
Clostridium botulinum. Preferably, the soy-based products are
water-soluble and derived from hydrolyzed soy, although as
previously discussed, insoluble soy products can also be used to
practice the present invention.
[0154] Any source of soy-based products may be used in accordance
with the present invention. Preferably, the hydrolyzed soy is
obtained from Quest International (Sheffield) under the tradename,
Hy-Soy or from DMV International Nutritionals (Fraser, N.Y.) under
the tradename, SE50MK. Soluble soy products can be also obtained
from a variety of sources including but not limited to Soy peptone
(Gibco) Bac-soytone (Difco), AMISOY (Quest), NZ soy (Quest), NZ soy
BL4, NZ soy BL7, and SE50MK (DMV International Nutritionals,
Fraser, N.Y.).
[0155] In another preferred embodiment of the present invention,
the medium used for fermentation of Clostridium botulinum is free
of animal by-products and comprises hydrolyzed soy, glucose, NaCl,
Na.sub.2HPO.sub.4, MgSO.sub.47H.sub.2O, KH.sub.2PO.sub.4,
L-cysteine, L-tyrosine, and powdered iron. As disclosed for the
seed medium, hydrolyzed soy can replace animal by-products in
fermentation medium. These animal by-products include BHI and
NZ-Case TT (enzymatically digested casein).
[0156] The concentration of Hy-Soy in the fermentation medium for
production of botulinum toxin preferably ranges between
approximately 10-100 g/L. Preferably, the concentration of Hy-Soy
ranges between approximately 20-60 g/L. Most preferably, the
concentration of Hy-Soy in the fermentation medium is approximately
35 g/L. For maximal production of botulinum toxin, particularly
preferred concentrations of components in the fermentation medium
are approximately 7.5 g/L, glucose; 5.0 g/L NaCl; 0.5 g/L
Na.sub.2HPO.sub.4; 175 mg/L KH.sub.2PO.sub.4; 50 mg/L
MgSO.sub.47H.sub.2O; 125 mg/L L-cysteine; and 125 mg/L L-tyrosine.
The amount of powdered iron used can range from 50 mg/L to 2000
mg/L. Preferably, the amount of powdered iron ranges between
approximately 100 mg/L and 1000 mg/L. Most preferably, the amount
of powdered iron used in fermentation media ranges between
approximately 200 mg/L and 600 mg/L.
[0157] For optimal levels of toxin production, the initial pH
(before autoclaving) of the soy-based fermentation media ranges
preferably between approximately 5.0 to 7.1. We found that pH
control improves botulinum toxin recovery. Preferably the initial
pH of the fermentation medium is about pH 7. As explained in
Example 7, we have found that a high yield of stable botulinum
toxin can be obtained if the pH is thereafter reduced to and
maintained between pH 5-5.5. As described for the seed medium, the
components of the fermentation medium, including glucose and iron,
are preferably autoclaved together for sterilization.
[0158] Preferably, a portion of the second stage seed medium used
for growth of Clostridium botulinum is used to inoculate the
fermentation medium. Fermentation occurs in an anaerobic chamber at
approximately 34.+-.1.degree. C. for approximately 7 to 9 days.
Bacterial growth can be monitored by measuring the optical density
(O.D.) of the medium. Fermentation preferably is stopped after cell
lysis has proceeded for at least 48 hours as determined by growth
measurement (optical density). As cells lyse, the O.D. of the
medium decreases.
[0159] In a preferred embodiment of the present invention, cultures
of Clostridium botulinum used for long-term storage of Clostridium
botulinum and inoculation of the seed medium are grown and
lyophilized in soy-milk prior to storage at 4.degree. C. Cultures
of Clostridium botulinum in animal milk lyophilized for storage can
also be used for the production of botulinum Toxin. However, to
maintain media that are substantially free of animal by-products
throughout the production of botulinum toxin, it is preferred that
the initial culture of Clostridium botulinum be preserved in soy
milk and not animal milk.
EXAMPLES
[0160] The following examples set forth specific methods
encompassed by the present invention and are not intended to limit
the scope of the invention. Unless explained otherwise in these
Examples "toxin" or "botulinum toxin" means a botulinum toxin type
A complex with a molecular weight of about 900 kDa. Our invention
is not limited to systems and method for purifying a botulinum
toxin type A complex with a molecular weight of about 900 kDa,
having ready applicability to the purification of 150 kDa, 300 kDa,
500 kDa and well as other molecular weight toxins, complexes and
botulinum toxin serotypes.
Example 1
Preparation of an Animal Product Free Seed Medium for Clostridium
Botulinum
[0161] A control seed medium can be prepared using the following
ingredients for each one 1 liter of medium: NaCl (5 g),
Bacto-peptone (10 g), glucose (10 g), BHI (to 1 liter), pH 8.1
(adjusted with 5 N NaOH).
[0162] A test (animal product free) seed medium can be prepared
using the following ingredients for each one 1 liter of medium:
NaCl (5 g), Soy-peptone (10 g), glucose (10 g), Hy-Soy (35 g/liter,
to make up 1 liter of media fluid), pH 8.1 (adjusted with 5 N
NaOH).
Example 2
[0163] Culturing Clostridium Botulinum in an Animal Product Free
Seed Medium
[0164] A lyophilized culture of the Clostridium botulinum can be
suspended in 1 ml of each of the control and test seed medium of
Example 1, divided (each seed media) into two tubes of which each
can contain 10 ml of the respective seed media, and then incubated
at 34.degree. C. for about 24-48 hours. One ml of culture can be
then used to inoculate a 125 ml DeLong Bellco Culture Flask
containing 40 ml of (the respective) seed media. The inoculated
culture can be incubated at 33.degree. C. .+-.1.degree. C. for 24
hours in a Coy Anaerobic Chamber (Coy Laboratory Products Inc.,
Grass Lake, Mich.).
Example 3
Preparation of an Animal Product Free Fermentation Media for
Clostridium Botulinum
[0165] A basal fermentation medium can be prepared using the
following ingredients for each two liters of medium: glucose (15
g), NaCl (10 g), NaH.sub.2PO.sub.4 (1 g), KH.sub.2PO.sub.4(0.350
g), MgSO.sub.47H.sub.2O (0.1 g), cysteine-HC (0.250 g),
tyrosine-HCl (0.250 g), powdered iron (1 g), ZnCl.sub.2 (0.250 g),
and MnCl.sub.2 (0.4 g).
[0166] A control fermentation medium can be prepared using the
following ingredients for each two liters of medium prepared: BHI
(500 ml; this corresponds to about 45.5 grams of dry weight beef
heart infusion), NZ-CaseTT (30 g), and basal medium (to 2 liters),
pH 6.8.
[0167] The basal fermentation medium can be prepared first and
adjusted to pH 6.8. The beef heart infusion (BHI) BHI can then be
prepared and it's pH adjusted to 0.8 with 5 N NaOH. The BHI can
then be added to the basal medium. Next the NZ-CaseTT can be
prepared. The NZ-Case TT is then added to the basal medium to which
the beef heart infusion has already been added, and dissolved by
addition of HCl. The pH can then be adjusted to 6.8 with 5 N NaOH.
This medium can then be separated into 8 ml portions into each of
sixteen 100 mm test tubes, following by autoclaving for 25 minutes
at 120.degree. C.
[0168] A test fermentation medium (animal product free) can be
prepared by substituting a test nitrogen source for the BHI present
in the control fermentation medium. Suitable test fermentation
medium nitrogen sources include: Hy-Soy (Quest), AMI-Soy (Quest),
NZ-Soy (Quest), NZ-Soy BL4 (Quest), NZ-Soy BL7 (Quest), Sheftone D
(Sheffield), SE50M (DMV), SE50 (DMV), SE %)MK (DMV), Soy Peptone
(Gibco), Bacto-Soyton (Difco), Nutrisoy 2207 (ADM), Bakes Nutrisoy
(ADM) Nutrisoy flour, Soybean meal, Bacto-Yeast Extract (Difco)
Yeast Extract (Gibco), Hy-Yest 412 (Quest), Hy-Yest 441 (Quest),
Hy-Yest 444 (Quest), Hy-Yest (455 (Quest) Bacto-Malt Extract
(Difco), Corn Steep, and Proflo (Traders).
[0169] The test fermentation medium can be prepared as set forth
above for a control fermentation medium except that BHI is excluded
and the relevant nitrogen source can be first adjusted to pH 6.8
with 3 N HCl or with 5 N NaOH. The media can be allocated to in 8
ml portions to sixteen 100 mm test tubes, followed by autoclaving
for 20-30 minutes at 120.degree. C.
Example 4
Growth of Clostridium Botulinum in an Animal Product Free
Fermentation Medium
[0170] A 40 .mu.l portion of the test seed medium culture (animal
product free) can be used to inoculate each 8 ml control or test
fermentation medium aliquot in an 8 ml 16.times.100 mm test tube.
The cultures can then be incubated at 33.+-.1.degree. C. for 24
hours. Tubes can then be incubated in an anaerobic chamber to allow
for growth of the bacterium. Each medium assay can be performed in
triplicate (i.e. can involve three independent inoculations of the
same medium), and can also include a non-inoculated control, which
can be used as the blank for the spectrophotometer). Growth (as
determined by optical density, OD) can be measured every 24 hours
with a Turner Spectrophotometer (Model 330) at 660 nm. Cultivation
should be stopped after cell lysis has lasted for about 48 hours
and botulinum toxin production can then be measured.
[0171] Additional experiments can be carried out with a Hy-Soy
fermentation medium containing the following ingredients for each
500 ml of the medium: Hy-Soy (17.5 g), glucose (3.75 g); NaCl (2.5
g); Na.sub.2HPO.sub.4 (0.25 g), MgSO.sub.47H.sub.2O (0.025 g),
KH.sub.2PO.sub.4 (0.0875 g), L-cysteine (0.0625 g), L-tyrosine
(0.0625 g), powdered iron (0.25 g), pH 6.8
Example 5
Determination of Botulinum Toxin Production by Clostridium
Botulinum Grown in an Animal Product Free Fermentation Medium
[0172] The cultured cells of Example 4 can be centrifuged, and the
pH of the supernatant then determined. The levels of botulinum
toxin in a given sample can be measured by adding a standard
antitoxin and measuring the elapsed time before flocculation. Both
Kf (the time required for flocculation to occur, in minutes) and Lf
(the limit of flocculation; equivalent to 1 international unit of
standard antitoxin, as established by flocculation) can be
determined. 4 ml of fermentation broth can be taken from each
fermentation tube for a given culture, and can be combined together
so that 12 ml total can be mixed in a 15 ml centrifuge tube. The
tubes can be centrifuged at 5000 rpm (3400 g) for 30 min at
4.degree. C. 1 ml aliquots of supernatant can be added to tubes
containing 0.1-0.6 ml of standard botulinum toxin antiserum, and
the tubes can be carefully shaken to mix their contents. The tubes
can then be placed in a water bath at 45.+-.1.degree. C. and the
initial time can be recorded. The tubes can be checked frequently,
and the time at which flocculation began can be recorded as Kf. The
concentration of toxin in the tube in which flocculation can be
first initiated can be designated LfFF. The concentration of toxin
in the tube in which flocculation can be initiated second can be
designated LfF.
[0173] Parallel fermentation, growth and toxin production assays
can be carried out for both of: (a) the control seed medium (used
to inoculate the control fermentation medium) and the control
fermentation medium, and; (2) the (animal product free) test seed
medium (used to inoculate the test fermentation medium) and the
(animal product free) test fermentation medium. Significantly, it
can be determined that the fermentation of Clostridium botulinum in
media free of animal products and inoculated from cultures also
free of animal products (with soy-base products replacing the
animal products) can result in an Lf.sub.toxin of approximately 50
or more. Minimally, Lf.sub.toxin equals approximately 10.
Preferably the Lf.sub.toxin is at least 20. Most preferably the
Lf.sub.toxin is greater than 50.
[0174] Additionally, it can be determined that various soy products
support Clostridium botulinum growth in fermentation media lacking
BHI. Thus soluble soy preparations can replace BHI for growth of
Clostridium botulinum. The best concentration can be 12.5 or 25
g/L. Hy-Soy (Sheffield) can give the highest growth. Insoluble soy
preparations can be less effective.
[0175] Furthermore, results can be obtained to show that Quest
Hy-Soy, DMV SE50MK, and Quest NZ-Soy can be effective soy products
in terms of their ability to replace BHI for Clostridium botulinum
growth. The results can reveal that the soy products (such as Quest
Hy-Soy, DMV SE50MK, and Quest NZ-Soy) that may be optimal for
growth can also be effective at replacing BHI for toxin production.
The best soy product for toxin production can be Quest Hy-Soy at
22.75 g/l. Higher concentrations of this product may produce better
growth but not improve toxin production. Similar results can, it is
proposed, be obtained with SE50MK, for which a higher concentration
may generate increased growth, but not increase toxin production.
NZ-Soy, on the other hand, may give higher growth and higher toxin
production at its higher concentration.
[0176] Finally, it can be determined that soy products can
effectively replace BHI as well as the NZ-CaseTT. Removal of
NZ-CaseTT from soy-based media can reduce growth of about 2-4 fold.
The best soy product for growth both in the presence and the
absence of NZ-CaseTT can be SE50MK. HY-Soy can replace both BHI and
NZ-CaseTT for toxin production. However, a longer fermentation
cycle of 1 or 2 days may be necessary. HY-Soy could replace both
BHI and NZ-CaseTT in media for toxin production. However, it can be
determined that yeast extracts can be inhibitory to toxin
production.
[0177] It can be determined that HY-Soy at 22.75 g/1 may completely
replace both BHI and HY-CaseTT for toxin production. Unlike the
effect on growth where 56.88 g/l HY-Soy can be best, 34.13 g/l
HY-Soy can be best for the toxin production phase.
[0178] Thus, it has surprisingly been determined if Hy-Soy or
[Hy-Soy+Hy-Yest] can replace BHI and Bacto-peptone in media for
seed growth of Clostridium botulinum. In addition, experiments can
be designed to determine the optimum concentrations of components
in seed media to produce the maximum levels of botulinum toxin
production by the Clostridium botulinum. Toxin production by
Clostridium botulinum grown in seed medium and fermentation medium
that is free of BHI and NZ-CaseTT can reach or exceed levels
attained in media containing BHI and NZ-CaseTT.
[0179] It can be determined that the optimum concentrations of
Hy-Soy or [Hy-Soy+Hy-Yest] for growth in the seed medium.
Experiments can confirm that Hy-Soy can replace BHI and
Bacto-peptone as the nitrogen source in seed medium for growth of
Clostridium botulinum and for production of botulinum toxin in the
subsequent fermentation phase. Also, Hy-Soy as nitrogen source in
the seed medium, as compared to Hy-Soy plus Hy-Yest, can produce
higher levels of botulinum toxin in the subsequent fermentation
step. The concentrations of Hy-Soy in seed medium that produce the
best levels of toxin range from approximately 62.5 g/L to 100
g/L.
[0180] Additional experiments can be designed to determine the
optimum concentrations of Hy-Soy in the seed medium for the maximum
production of botulinum toxin by Clostridium botulinum by
fermentation. Thus, 30 g, 50 g, 75 g and 100 g of Hy-Soy in the
seed medium can all resulted in production of botulinum toxin by
fermentation of Clostridium botulinum and this is comparable or
exceeds levels of botulinum toxin made in seed medium containing
BHI and Bacto-peptone as a nitrogen source.
[0181] It can be found that a concentration of 100 g/L Hy-Soy in
the seed medium resulted in the highest levels of toxin production
in the subsequent fermentation step. In addition, the data indicate
that seed step-1 of Hy-Soy seed medium produced greater growth
after 48 hours than after 24 hours.
Example 6
Non-APF Process for Obtaining a Botulinum Toxin
[0182] A Clostridial toxin was obtained by fermentation of a
Clostridium botulinum bacterium. Thus, a modified Schantz (non-APF)
process was carried out to obtain highly potent and highly purified
Clostridium botulinum toxin (i.e. bulk toxin). Botulinum toxin is
also referred to as "toxin". A modified Schantz (non-APF) process
can provide a high yield of botulinum toxin. Both Schantz and
modified Schantz processes use casein in all the fermentation
media.
[0183] Stock Culture Preparation
[0184] Various Clostridial bacteria are available from the American
Type Culture Collection (ATCC), Manassas, Va. Alternately, a
Clostridium botulinum cell bank vial can be prepared by isolating
Clostridium botulinum from various sources, including soil or by
deep sampling (at anaerobic or at quasi-anaerobic locations) of
putrefying animal carcasses. Commonly, Clostridium botulinum can be
obtained from a sample of a physiological fluid (i.e. a wound swap
from a patient with wound botulism) of a patient diagnosed with
botulism. The top half of FIG. 1 summarizes the non-APF process
used for preparation of a cell bank vial, and for the culture and
fermentation of a botulinum toxin.
[0185] The Clostridium botulinum obtained from a natural or patient
source is cultured on blood agar plates, followed by inoculation of
high growth colonies into a cell bank vial medium. The cell bank
vial medium used for Clostridium botulinum was a cooked meat medium
which contains chopped fresh beef. Actively growing cultures were
mixed with glycerol to prepare a cell bank vial (i.e. a stock
culture) of the Clostridium botulinum bacterium which was frozen
for later use.
[0186] Seed Cultivations
[0187] A Clostridium botulinum cell bank vial was thawed at room
temperature, followed by four cultivation steps. (1) To select
colonies with a suitable morphology, aliquots from the thawed cell
bank vial were cultivated by streaking the bacterium on pre-reduced
Columbia blood agar plates and anaerobically incubating for 3048
hours at 34.degree. C..+-.1.degree.. (2) Selected colonies were
then inoculated into test tubes containing a casein growth medium
for 6-12 hours at 34.degree. C. The contents of the tube with the
most rapid growth and highest density (growth selection step) were
then further cultivated through two step-up anaerobic incubations:
(3) a first 12-30 hour incubation at 34.degree. C. in a one liter
seed cultivation bottle, followed by (4) a second cultivation in a
25 liter seed fermenter containing a casein growth medium for 6-16
hours at 35.degree. C. These two step-up cultivations were carried
out in a nutritive media containing 2% casein hydrolysate (a casein
[milk protein] digest), 1% yeast extract and 1% glucose (dextrose)
in water at pH 7.3.
[0188] Fermentation
[0189] The step-up cultivations were followed by a further
incubation for 60-96 hours at 35.degree. C. in a commercial scale
(i.e. 115 liter) fermenter in a casein containing medium under a
controlled anaerobic atmosphere. Growth of the bacterium is usually
complete after 24 to 36 hours, and during the 60-96 hour
fermentation most of the cells undergo lysis and release botulinum
toxin. Control of the fermentation medium pH is not required in a
Schantz or modified Schantz process. It is believed that toxin is
liberated by cell lysis and activated by proteases present in the
culture broth. Optionally, a filtration of this culture medium
using a single layer depth filter to remove gross impurities (i.e.
whole and ruptured cells) can be prepared to obtain a clear
solution referred to a clarified culture.
[0190] Harvest
[0191] Harvest of toxin from the clarified culture was accomplished
by lowering the pH of the clarified culture to pH 3.5 with 3M
sulfuric acid (acidification) to precipitate the raw toxin at
20.degree. C. The raw toxin was then concentrated (to achieve a
volume reduction) by ultramicrofiltration (microfiltration)
(referred to as MF or UF) followed by diafiltration (DF). A 0.1
.mu.m filter can be used for the microfiltration step.
[0192] Purification
[0193] The harvested crude or raw toxin was then transferred to a
digestion vessel and stabilized by addition of the protease
inhibitor benzamidine hydrochloride. DNase and RNase were added to
digest (hydrolyze) nucleic acids. Hydrolyzed nucleic acids and low
molecular weight impurities were then removed by further UF and DF
steps. The toxin was then extracted with pH 6.0 phosphate buffer
and cell debris removed by clarification. Next three sequential
precipitation steps (cold ethanol, hydrochloric acid and ammonia
sulfate precipitations) were carried out. The purified botulinum
neurotoxin complex (bulk toxin) was stored as a suspension in a
sodium phosphate/ammonium sulphate buffer at 2.degree. to 8.degree.
C.
[0194] The entire harvesting and purification steps can take about
two weeks to accomplish. The resulting bulk toxin was a high
quality crystalline 900 kD botulinum toxin type A complex made from
the Hall A strain of Clostridium botulinum with a specific potency
of .gtoreq.3.times.10.sup.7 U/mg, an A.sub.260/A.sub.278 of less
than 0.60 and a distinct pattern of banding on gel electrophoresis,
and suitable for use for the compounding of a botulinum toxin
pharmaceutical composition.
[0195] Compounding can encompass a many fold dilution of the bulk
toxin, mixing with one or more excipients (such as albumin [such as
a human serum albumin or a recombinant human albumin] and sodium
chloride) to thereby form a toxin composition, and preparation of a
storage and shipment stable form of the toxin composition, as by
lyophilizing, freeze drying or vacuum drying the composition.
[0196] The purified botulinum toxin complex obtained from a Schantz
or modified Schantz process can be eluted from an ion exchange
column in a pH 7-8 buffer to disassociate the non toxin complex
proteins from the botulinum toxin molecule, thereby providing
(depending upon the type of Clostridium botulinum bacterium
fermented) pure botulinum toxin type A with an approximately 150 kD
molecular weight, and a specific potency of 1-2.times.10.sup.8
LD.sub.50 U/mg or greater; or purified botulinum toxin type B with
an approximately 156 kD molecular weight and a specific potency of
1-2.times.10.sup.8 LD.sub.50 U/mg or greater, or purified botulinum
toxin type F with an approximately 155 kD molecular weight and a
specific potency of 1-2.times.10.sup.7 LD.sub.50 U/mg or
greater.
[0197] As set forth supra, in one aspect our invention eliminates
the harvest purification steps set forth in this Example 6 carried
out upon clarified culture, including elimination of use of the
animal derived products, such as RNase and DNase.
Example 7
APF Media and Process for Obtaining a Botulinum Toxin
[0198] This Example 7 sets forth an APF process carried out to
obtain highly potent and highly purified Clostridium botulinum
toxin type A (i.e. bulk toxin). The process can be used with other
botulinum toxin serotypes.
[0199] Stock Culture Preparation
[0200] As set forth in Example 6, Clostridial botulinum can be
obtained from the ATCC, from various sources in nature or from a
botulism patient. The bottom half of FIG. 1 summarizes the APF
process used for preparation of a cell bank vial, and for the
culture and fermentation of a botulinum toxin. APF cell bank vials
were prepared by culturing Clostridium botulinum on plant agar
plates. The plant agar plates were made by mixing the soy
derivative HySoy (Quest) with a yeast extract and glucose in a
3:1:1 (weight percent) ratio with agar and allowing setting. Other
commercially available APF agar plates or dehydrated powder for
making the plates were also found to be suitable. Selected high
growth colonies were then inoculated into an APF cell bank vial
medium. The APF cell bank vial medium used comprised hydrolyzed soy
protein, yeast extract (no animal product was used in either the
cultivation of the yeast or in the process for preparation of the
yeast extract made therefrom) and glucose in the same 3:1:1 ratio.
Other nutrient ratios (i.e. 6:1:1, 6:0:1 and 6:3:1 were also found
to be suitable). The hydrolyzed soy (HySoy) and yeast extract
(HyYest) concentrates used were obtained from Quest International.
The Clostridium botulinum culture in the APF medium was combined
with glycerol, aliquoted to cryovials and frozen for later use. The
APF media developed can be used to store the Clostridial botulinum
bacteria for a period of one year or longer without loss of
viability. These frozen culture and glycerol mixtures in cryovials
are the APF cell bank vials.
[0201] Seed Cultivations
[0202] An APF cell bank vial was thawed at room temperature,
followed by a single cultivation step: a one liter seed culture
bottle was then inoculated directly (i.e. without an intervening
blood agar culture or tube growth steps) with the APF cell bank
vial contents using the same APF medium (the APF cell bank vial
[storage] medium can be different from the APF fermentation
[growth] medium) and maintained at 35.degree. C. for 15 to 24
hours, with an initial medium pH of 7.0 in an anaerobic (nitrogen)
atmosphere.
[0203] Fermentation
[0204] Next the seed bottle culture was transferred to a commercial
scale 10 liter production fermenter containing the APF medium
(hydrolyzed soy protein, yeast extract and 1% glucose) maintained
at 35.degree. C. for 52-72 hours, with an initial medium pH of 7.0,
in an anaerobic (nitrogen) atmosphere. Approximately 15 hours after
commencement of the fermentation (the culture pH had naturally
decreased to below 6.0), a pH control program at range of pH
5.0-5.5 was initiated by adding HCl to the culture. It was found
that it was necessary to control the pH of the APF fermentation
medium within this narrow pH range in order to obtain an acceptable
yield of active botulinum toxin. Thus, it was found that this pH
control to between pH 5.0-5.5 substantially prevented degradation
and loss of potency of the botulinum toxin. It is believed that
during the fermentation most of the cells undergo lysis and release
botulinum toxin and that toxin liberated by cell lysis is activated
by proteases present in the culture broth. Filtration of this
culture medium using a single layer depth filter removes gross
impurities (i.e. whole and ruptured cells) and results in a clear
solution referred to a clarified culture.
[0205] Harvest
[0206] Harvest of botulinum toxin can then proceed as in Example 6
(i.e. sulfuric acid precipitation, followed by concentrated by
microfiltration followed by diafiltration).
[0207] Purification
[0208] Purification of the toxin can then proceed as set forth in
Example 6: i.e. addition of benzamidine hydrochloride, and DNase
and RNase, sulfuric acid precipitation, cold ethanol precipitation,
phosphate buffer extraction, hydrochloric acid precipitation,
phosphate buffer extraction and bulk toxin storage.
[0209] As an alternative to the Example 6 harvest and purification
process, a column chromatography process of the present invention
can be carried out.
[0210] The resulting bulk toxin is a high quality crystalline 900
kD botulinum toxin type A complex made from the Hall A strain of
Clostridium botulinum with a specific potency of
.gtoreq.3.times.10.sup.7 U/mg, an A.sub.260/A.sub.278 of less than
0.60 and a distinct pattern of banding on gel electrophoresis, and
suitable for use for the compounding of a botulinum toxin
pharmaceutical composition. Thus, this APF process for a botulinum
toxin can generate high quality toxin.
[0211] The purified botulinum toxin complex obtained from an APF
process can be passed through and eluted from an ion exchange
column in a pH 7-8 buffer to disassociate the non toxin complex
proteins from the botulinum toxin molecule, thereby providing
(depending upon the serotype of Clostridium botulinum bacterium
fermented) botulinum toxin with an approximately 150 kD molecular
weight, and a specific potency of 1-2.times.10.sup.8 LD.sub.50 U/mg
or greater; or purified botulinum toxin type B with an
approximately 156 kD molecular weight and a specific potency of
1-2.times.10.sup.8 LD.sub.50 U/mg or greater, or purified botulinum
toxin type F with an approximately 155 kD molecular weight and a
specific potency of 1-2.times.10.sup.7 LD.sub.50 U/mg or greater.
For example, by use of our APF medium we were able to obtain a
botulinum toxin type A complex with a specific potency of
1.02.times.10.sup.8 LD.sub.50 U/mg of the botulinum toxin.
[0212] In this Example 7 APF media with either 1% by wt or 2% by wt
glucose were used (note that 1% glucose means 1 g of glucose per
100 ml of the culture medium and 2% glucose means 2 g of glucose
were present for each 100 ml of the culture medium) and it was
determined that maximal bacterium growth (as determined by peak
optical density [optical density was measured at 600 nm] of the
culture) occurred after about 20 hours of fermentation in the 1%
glucose APF medium vs after about 40 hours of fermentation in the
2% glucose APF medium, but that the peak optical densities did not
differ significantly as the glucose content of the media was so
varied. It was believed that cell autolysis and toxin release
resulted in a maximal amount of active botulinum toxin in the 1%
glucose APF media (as determined by a SNAP-25 assay for active
toxin) after about 55 hours of fermentation, but that with the 2%
glucose APF media the amount of active botulinum toxin present in
the medium at a later time (as determined by a SNAP-25 assay for
active toxin) and was still increasing after 65 hours of
fermentation. Thus, a more rapid release of botulinum toxin
occurred with use of the lower (1%) glucose APF medium amount
present, indicating that a more efficient toxin production process
(i.e. more amount of toxin obtained per unit of time) can be
carried out with use of the lower (1%) glucose APF medium.
[0213] As shown by FIG. 1, it was also determined that optimal
parameters for production of botulinum toxin in an APF medium were
the combination of the following parameters: (1) about 6% by weight
of a hydrolyzed soy concentration ("HySoy Conc." in FIG. 1) in the
APF fermentation medium. 6% soy means 6 g of the soy protein per
100 ml of the culture medium; (2) 0% to 3% yeast extract
concentrate ("YE Conc." In FIG. 1) in the APF fermentation medium;
(3) 50-72 hours of fermentation at a temperature of 33-35.degree.
C. under anaerobic (nitrogen atmosphere) conditions; (4) pH of the
fermentation medium maintained between about pH 5.0 to 5.5
throughout the fermentation period after the initial cell growth,
and (5) 1 wt % glucose in the APF fermentation medium.
[0214] Thus, as shown by FIG. 1 as more protein is present in the
APF medium (as the total amount of HySoy and YE) the pH of the
medium tends to increase with resulting lower toxin stability and
that when the pH was lowered with the same total protein nutrient
content in the medium, toxin production yield increased
dramatically. In the non-APF process the total protein content is
lower so that pH does not tend to rise and therefore there is no
elevated pH to have a deleterious effect on toxin production. FIG.
1 shows that there was consistently more activity (as determined by
the MLD50 and SNAP-25 assays) when the pH of the medium was
controlled to within a narrow range of about 5.3 to 5.5. FIG. 1
also shows that the highest toxin yield (as determined by the SNAP
25 assay) was obtained with a medium which comprised 6% hydrolyzed
soy and 1% yeast extract. FIG. 8 shows that when the yeast-glucose
nutrients were both held at 1 wt % of the fermentation medium, that
cell lysis between 68% to 100% and toxin potency of up to about 38
MLD.times.10.sup.5/mL of toxin was obtained as the soy protein was
varied in the fermentation medium was varied from 1 wt % to 6 wt
%.
[0215] The SNAP-25 assay used was an ELISA based method to measure
SNAP-25 proteolytic activity of the botulinum toxin. SNAP-25 is an
abbreviation for synaptosome associated protein of 25 kDa molecular
weight. SNAP-25 is a 206 amino acid plasma membrane protein
involved in neuronal exocytosis. The assay is based on the method
disclosed in Ekong T., et al., Recombinant SNAP-25 is an effective
substrate for Clostridium botulinum type A toxin endopeptidase
activity in vitro, Microbiology (1997), vol 143, pages 3337-3347.
The assay uses a truncated SNAP-25 protein (the 206 amino acid
residue peptide) bound to polystyrene 96 well microtiter plates and
a monoclonal antibody that recognizes the cleaved product (a 197
amino acid residue peptide) which is made by enzymatic hydrolysis
between amino acids 197 and 198 of the SNAP-25 by reduced botulinum
toxin type A. The monoclonal antibody bound to the cleaved product
is then detected with a secondary antibody (goat anti-mouse IgG
conjugated to horseradish peroxidase [HRP)], which produces a color
change in the presence of a chromogenic substrate (TMB).
[0216] The MLD50 (mouse 50% lethal dose) assay is a method for
measuring the potency of a botulinum toxin by intraperitoneal
injection of the botulinum toxin into female mice (about four weeks
old) weighing 17-22 grams each at the start of the assay. Each
mouse is held in a supine position with its head tilted down and is
injected intraperitoneally into the lower right abdomen at an angle
of about 30 degrees using a 25 to 27 gauge 3/8'' to 5/8'' needle
with one of several serial dilutions of the botulinum toxin in
saline. The death rates over the ensuing 72 hours for each dilution
are recorded. The dilutions are prepared so that the most
concentrated dilution produces a death rate of at least 80% of the
mice injected, and the least concentration dilution produces a
death rate no greater than 20% of the mice injected. There must be
a minimum of four dilutions that fall within the monotone
decreasing range of the death rates. The monotone decreasing range
commences with a death rate of no less than 80%. Within the four or
more monotone decreasing rates, the two largest and the two
smallest rates must be decreasing (i.e. not equivalent). The
dilution at which 50% of the mice die within the three day post
injection observation period is defined as a dilution which
comprises one unit (1 U) of the botulinum toxin.
[0217] Significantly, the APF process of this Example 7 differs
from the Example 6 non-APF process, by at least: (1) replacing the
cell bank vial cooked meat medium with an APF medium; (2)
eliminating the blood agar colony selection step; (3) eliminating
the subsequent casein medium based tube growth step, and; (4)
replacing the non-APF fermentation media with APF media
throughout.
[0218] FIG. 2 presents a summary of the differences between an
industrial scale (non-APF) Schantz process (Example 6 and the
industrial scale APF process of Example 7, through the cell bank
creation, culture and fermentation steps. FIG. 2 omits the harvest
and purification steps.
[0219] The APF media can be used to select for Clostridium
botulinum bacteria. Thus, concurrent practice of the Examples 6 and
7 initial culture steps permits isolation and growth of a
Clostridium botulinum culture with characteristics conducive to
growth and production of botulinum toxins in or on an APF medium.
The transfer of Clostridium botulinum bacteria from a non-APF
medium to an APF medium enriches for and selects for bacteria that
can either adapt to the new environment or through selective die
off of bacteria that cannot grow and produce in the new
environment.
Example 8
APF Chromatographic Systems and Methods for Purifying a Botulinum
Toxin
[0220] The chemicals used in the experiments set forth in Examples
8 and following included:
10N NaOH (Mallinckrodt, VWR Cat # MKH38505)
Acetic Acid, USP/FCC Grade, 99.5-100.5% (J. T. Baker, Cat #
JT9522-2)
Ammonium Sulfate, Ultrapure, 99% (ICN, Cat# IC808211)
Citric Acid, USP/FCC Grade, 99.5-100.5% (J. T. Baker,
Cat#JT0119-1)
[0221] Ethanol, anhydrous, denatured (JT Baker, Cat # 9299-1)
Hydrochloric acid, NF/FCC Grade, 36.5-38%-Mallinckrodt-MK2612-14
Phosphoric acid, NF/FCC, 85%-88% (Mallinckrodt, Cat # MK278814)
Sodium acetate trihydrate, 99%-101%, USP/FCC (Mallinckrodt, Cat #
MK735602) Sodium chloride, USP/FCC Grade, 99.0-101.0 (Mallinckrodt,
Cat # MK753204) Sodium citrate, USP/FCC Grade, 99.0-100.5% (J. T.
Baker, Cat # JT3650-1) Sodium hydroxide, NF/FCC Grade,
95.0-100.5%-Mallinckrodt-MK768004 Sodium phosphate, dibasic
Heptahydrate, USP (Mallinckrodt, Cat # MK789604) Sodium phosphate,
monobasic monohydrate, USP/FCC (Mallinckrodt, Cat # MK786812)
[0222] The chromatography resins use in the experiments below
included:
[0223] Bakerbond ABx Prepscale (JT Baker, Cat # 7269-02)
[0224] Butyl Sepharose FF (GE Healthcare, Cat # 17-0980-02)
[0225] Ceramic Hydroxyapatite, Type I (Bio-Rad, Cat # 158-4000)
[0226] Ceramic Hydroxyapatite, Type II (Bio-Rad, Cat# 157-4200)
[0227] HiTrap HIC Selection Kit (GE Healthcare, Cat #
17-1349-01)
[0228] HiTrap IEX Selection Kit (GE Healthcare, Cat #
17-6002-33)
[0229] MEP Hypercel (Ciphergen, sample)
[0230] SP Sepharose HP (GE Healthcare, Cat # 17-1087-03)
[0231] The equipment and accessories used is the experiments below
included: AKTA Purifier and AKTA FPLC Chromatography System
(Amersham Biosciences)
Bottle-top 0.22 .mu.m vacuum sterile filter (Nalgene) Labscale TFF
system and Pellicon XL50 with Biomax 100 membrane (Millipore) (this
is the ultrafiltration equipment). Masterflex L/S pump Model
#77201-62 (Cole-Parmer)
Pellicon 2 Mini Holder (Millipore)
[0232] XK and HR columns (GE Healthcare)
[0233] The buffers used in our experiments are listed in Table
2.
TABLE-US-00003 TABLE 2 Buffers used in the APF purification process
Purification Steps Buffers used Butyl Sepharose 1. 50 mM NaPi, 4M
NaCl, pH 6.0 FF Chromatography 2. 50 mM NaPi, 2M NaCl, pH 6.0 3. 50
mM NaPi, 1M NaCl, pH 6.0 4. 50 mM NaPi, pH6,0 SP Sepharose HP 5. 20
mM Na citrate, pH 4.0 Chromatography 6. 20 mM Na citrate, 300 mM
NaCl, pH 4.0 7. 20 mM Na citrate, 400 mM NaCl, pH 4.0 8. 20 mM Na
citrate, 1M NaCl, pH 4.0 Post Purification Steps Solutions used
Post-column processes 9. 50 mM NaAc, pH 4.0 10. 3.5M ammonium
sulfate Miscellaneous 11. 0.1N NaOH 12. 1N NaOH
[0234] In Table 2: buffers 1 and 2 were used to wash impurities off
the column; buffers 3 and 4 was used to elute bound toxin from the
column; buffer 5 was used to dilute the eluent from the Butyl
column; buffer 6 was used to wash impurities off the column;
buffers 7 and 8 were used to elute bound toxin from the column;
buffer 8 was the UF/DF dialysis buffer; solution 9 was used to
precipitate toxin, and solutions 10 and 11 were used to inactivate
(clean) any toxin remaining in the columns after use.
Example 9
Selection of Preferred Chromatography Columns for use in an APF
Column Chromatographic Botulinum Toxin Purification (Capture Step)
Process
[0235] This experiment established preferred chromatography columns
and techniques for initial purification of a botulinum toxin type A
complex from attendant impurities in a fermentation medium.
[0236] Feed Materials
[0237] Both a filtered cell culture (clarified culture) obtained
from an APF process fermentation and an extract thereof prepared by
hydrochloric acid precipitation were assessed as chromatography
column feed materials. It was found that direct loading of the
clarified culture onto a column prevented toxin precipitation and
that a clarified culture feed material was much easier to handle
and validate. On the other hand, use as the feed material of a
clarified culture extract prepared by acid precipitation removed
additional impurities and provided virus inactivation. With regard
to the characteristics of process robustness, a clarified culture
was determined to be the preferred feed material, as opposed to use
of a hydrochloric acid precipitation preparation as the bulk
botulinum toxin complex chromatography resin feed material. Hence,
clarified culture was the preferred feed material.
[0238] Our studies showed that as the pH was lowered proteins (i.e.
the botulinum toxin complex) started to precipitate at about pH 5,
that small amounts of toxin was extracted (as most had precipitated
out) at about pH 4.0, and that essentially all of the toxin had
precipitation out of the solution at between pH 3.5 to 3.8. On the
other hand, we found (based for example on SDS-PAGE and Western
blotting) that most impurities were co-extracted with the botulinum
toxin at a pH of 6.8. Hence, a preferred feed liquid pH for
carrying out our purification process invention was between about
pH 5-6.8, with a more preferred pH being about pH 5.5 for
extraction, that is separation of the botulinum toxin from
attendant impurities.
[0239] Capture Step
[0240] For the capture step botulinum toxin type A (Hall strain)
cell culture filtrates were incubated with a number of
chromatography resins (see below) under the manufacturer specified
conditions for use of each particular column.
[0241] After washing the columns, the column bound proteins were
eluted with the specified elution buffers. All eluted fractions
were collected and analyzed by SDS-PAGE. The results obtained
(Table 3) were confirmed by chromatography using 1 ml HiTrap or
HR5/5 columns.
TABLE-US-00004 TABLE 3 Summary of Capture Step Results Separation
Toxin in Toxin in Separation Technique Resin Flowthru Eluate
Observed Hydrophobic Phenyl FF (HS) - + + Interaction Octyl FF - +
+ Butyl FF - + + Ion Exchange Q FF + - + SP FF + - - Mixed Mode HA
Type I + - - HA Type II + - - Abx + - - Hydrophobic MEP - + -
Charge-Induction Immobilized Chelating FF + - - Metal-ion
Affinity
[0242] This experiment clearly showed that the desired separation
of the botulinum toxin from other substances present was best
achieved by use of hydrophobic type column chromatography. Thus, we
found that the botulinum toxin bound to hydrophobic columns, but
that it did not bind to an ion exchange column, such as the Q
Sepharose FF column.
[0243] Among the hydrophobic columns evaluated, the weakly
hydrophobic Butyl Sepharose FF gave the best resolution. Therefore,
either Butyl Sepharose FF in binding mode or Q Sepharose FF in
flowthru mode provided a preferred botulinum toxin capturing
step.
[0244] Thus, we determined that an efficient capturing step can be
carried out using a hydrophobic column, such as the Butyl Sepharose
column chromatography. Presumably, the toxin binds to the Butyl
column via a hydrophobic interaction. Prior to this experiment it
was unknown that a botulinum toxin complex could be purified toxin
directly from clarified culture using a hydrophobic chromatography
column. We found that the Butyl Sepharose Fast Flow column has high
binding capacity, allows fast flow rate with low back pressure and
is therefore suitable for the capturing step that requires fast
removal of impurities.
Example 10
Four Column APF Chromatographic System and Process for Purifying a
Botulinum Toxin Complex
[0245] Intermediate and Polishing Purification Steps
[0246] Additional (intermediate and polishing) toxin purification
steps were carried out using the toxin-containing fractions
obtained from the preferred Q and Butyl columns of Example 9.
[0247] Three types of chromatography columns were found effective
for such further purification of the botulinum toxin complex. A
Hydroxyapatite (HA) type I column was a preferred column we used
because it showed separation, but some toxin was found in the flow
through. Gel filtration with a Superdex 200 column was a more
preferred column to use because it permitted purification of the
900 kDa botulinum toxin complex from the impurities, but a minor
impurity band was still present on SDS-PAGE.
[0248] A most preferred column was a SP Sepharose HP column which
we found to separate the botulinum toxin from impurities with very
good resolution. The botulinum toxin was pure after SP Sepharose HP
chromatography, based on analysis by SDS-PAGE.
TABLE-US-00005 TABLE 4 Summary of Column Chromatography
Purification Steps Separation technique Resin Summary Mixed mode
Hydroxyapatite Toxin in flow through mode, separated type I some
impurities. Gel Superdex 200 Partially purified toxin, difficult to
filtration scale-up, low productivity. Ion SP Sepharose HP High
resolution separation, pure toxin exchange obtained.
[0249] Based on the results of Examples 8 and 9, and as shown by
Table 4, the following four column chromatography purification
process was developed:
1. use of a Q Sepharose FF column for initial purification of a
clarified culture. In this step impurities bound to the column and
the toxin flowed through the column; 2. the eluent from the Q
Sepharose FF column step 1 was then passed through a Butyl
Sepharose FF column. The toxin bound to the column and was eluted
off with a suitable buffer; 3. the eluent from the Butyl Sepharose
FF was then passed through a Hydroxyapatite type I column.
Impurities bound to the column and the toxin flowed through the
column; 4. the eluent from the Hydroxyapatite type I was then
passed through an SP Sepharose column. The toxin bound to the
column and was eluted off with a suitable buffer.
[0250] This four column toxin purification process can be
summarized as:
APF clarified culture=>Q(flow
through)=>Butyl(binding)=>HA(flow
through)=>SP(binding)=>purified toxin complex
[0251] This four column bulk botulinum toxin complex process
allowed direct loading of filtered culture supernatant onto the Q
column (step 1). The flow through was supplemented with ammonium
sulfate to 0.8M before the second step of loading onto the Butyl
column. For the third step, the butyl eluate was loaded onto the HA
column directly, while the flow through of the HA was diluted 4
times with deionized water and the pH was adjusted to 4.0 before
loading onto the SP column for the fourth column step. This four
column process required minimal sample handling at each step, and
ensured that the toxin was exposed to mild buffering conditions
throughout the four steps of this purification process.
[0252] A scale up of the four column purification process set forth
above was used carried out upon 680 ml of filtered culture
supernatant obtained from an APF botulinum toxin type A
fermentation process. The results (see Table 5) show that this four
column process resulted in highly in a high yield of highly
purified botulinum toxin type A complex.
TABLE-US-00006 TABLE 5 Results of a Scale Up Purification using the
Four Column Purification process. Toxin yield ~30 mg per L culture
based on UV and Hc-ELISA. Toxin purity >98%, monodisperse, 900
kDa complex based on SEC- HPLC and LS. Pure on SDS-PAGE, western
blotting conforms to standard. Toxin potency 3-5 .times. 10.sup.7
MLD.sub.50 units per mg based on mouse toxicity assays.
Example 11
Additional Multi-Column APF Chromatography Processes for Purifying
a Botulinum Toxin Complex
[0253] Using the same procedures set forth in Examples 9 and 10
additional column combinations were evaluated. It was determined
that each of the following four additional column combinations
provided APF methods for obtaining highly purified botulinum toxin
complex, as determined by SDS-PAGE.
1. Q (flow through)=>Butyl=>SP 2. Butyl=>Q or HA (flow
through)=>SP 3. Butyl=>SP=>Q or HA (flow through)
4. Butyl=>SP
[0254] The purified toxins were further analyzed by SEC-HPLC with
light scattering, capillary electrophoresis, residual DNA assay,
Hc-ELISA, and MLD50. No significant differences were found among
the toxins from the four different processes set forth above. The
results are summarized in Table 6.
TABLE-US-00007 TABLE 6 Quality summary of toxin samples purified by
different APF processes 1.4. above. SEC-HPLC/LS Purity >99%,
purer than BCC2030, but less homogeneous than BCC2030. Capillary
Identical to one another, similar to 19P and 20P eletrophoresis
Research Grade APF Toxin, but slightly different from BCC2030.
Picogreen DNA 2-6 ng/ml, significantly lower than BCC2030. assay
Mouse toxicity Toxin potency 3.1-4.8 .times. 10.sup.7 MLD.sub.50
units/mg toxin assay, Hc- (by UV), or 3.8-12 .times. 10.sup.7
MLD.sub.50 units/mg toxin ELISA (by Hc-ELISA). Silver staining
Identical to one another. SDS-PAGE
Example 12
Two Column APF Chromatography Process and System for Purifying a
Botulinum Toxin Complex
[0255] Based on the results obtained in Example 11a two column
(Butyl =>SP) column chromatography process was selected for
further development.
[0256] Optimization of the First Step: Butyl Sepharose FF Toxin
Capture
[0257] Feed: Feed is to the clarified culture loaded on the column.
Since ammonium sulfate can affect the buffer pH, the use of NaCl to
replace ammonium sulfate in Butyl column was evaluated. We found
that addition of NaCl to the feed sufficient to 2M NaCl allowed the
botulinum toxin complex to bind to the butyl column. Subsequently,
we determined that feed at a 4M NaCl increased the binding of
botulinum toxin complex to the Butyl column, such that the yield of
toxin from the Butyl column was increased by 30% to 50%, as
determined by Hc-ELISA, as compared to use of feed at 2M NaCl.
[0258] The addition of NaCl to the clarified culture (the feed)
caused a small pH shift. However, the acceptable feed pH was
established between pH 5 and pH 6 and the final pH of the feed
after NaCl addition was within pH 5 and pH 6. Hence the preferred
feed to use in this first step of a two column purification process
has a 4M NaCl concentration and is at pH 5-6. Solid NaCl was added
to the clarified culture directly to obtain the 4M NaCl
concentration and this feed was then added to the Butyl column. The
bound toxin was eluted from the column using a 1M NaCl elution
buffer.
[0259] It was surprising that most of impurity proteins could be
washed away from the column and most of toxin bound to the column
could be eluted with a 1M NaCl buffer because column purification
processes typically consist of 3 or more columns, except for an
affinity column process. We determined that this butyl column is
unique as it has the ability to remove many of the impurity
proteins. Thus, after use of this column the botulinum toxin
complex purity was approximately 50%.
[0260] A wash step was then carried out to remove impurities from a
column. The impurities in the column came from the clarified
culture feed (containing 4M NaCl) used. The optimized washing steps
were: 1) Wash #1: 5CV of 50 mM NaPi, 4M NaCl, pH 6.0, and 2) Wash
#2: 12CV of 50 mM NaPi, 2M NaCl, pH 6.0. When 12CV and 5CV were
compared, it was found that 5CV is not sufficient in removing the
impurities, while the wash is to remove impurities after loading
the clarified culture in this case.
[0261] Elution (to remove toxin bound to a column). Toxin elution
with 1.2M, 1.0M and 0.8M NaCl were evaluated. It was chosen to
elute toxin with 1M NaCl in 50 mM NaPi, pH 6.0, based on toxin
recovery and impurity removal.
[0262] Low salt wash: After elution, the column was further washed
with 50 mM NaPi, pH 6.0 to remove residual impurities bound to the
column for the characterization of purification process.
[0263] Cleaning: the column was cleaned with 3CV of 0.1N NaOH to
inactivate any residual toxin before the disposal of used
resin.
[0264] Running flow rate: The typical flow rate was 100 cm/h. The
loading flow rate was between 90 cm/h and 120 cm/h depending on the
back pressure.
[0265] Loading capacity: Typical loading capacity was 12.7 ml
culture per ml bed, or at production scale, 10 L culture for 785 ml
resin bed (BPG 100 column at 10 cm bed height).
[0266] Bed height: All columns were packed with standard 10 cm bed
height.
[0267] Optimization of the Second Step: SP Sepharose HP
Purification
[0268] Feed conditioning: The Butyl eluate was diluted 5 times with
20 mM Na citrate buffer, pH 4.0, and the feed pH was adjusted to
4.0. The five times dilution step was carried out to condition the
hydrophobic interaction chromatography eluent for use in ion
exchange chromatography. We found that the optimal feed pH for best
toxin recovery was within the range of pH 4.0.+-.0.2.
[0269] Wash step: After loading, the column was washed with 1) 5CV
of 20 mM Na citrate, pH 4.0, followed by 2) 3-5CV of 20 mM Na
citrate, 300 mM NaCl, pH 4.0 to remove impurities before the
elution of bound toxin.
[0270] Elution step: The toxin was eluted with 20 mM Na citrate,
400 mM NaCl, pH 4.0.
[0271] High salt washing step: After elution, the column was
further washed with 20 mM Na citrate, 1M NaCl, pH 4.0 to remove
strongly bound impurities.
[0272] Column cleaning: The column was cleaned with .about.3CV of
0.1N NaOH to inactivate residual toxin before the disposal of used
resin.
[0273] Flow rate: The typical flow rate was 100 cm/h.
[0274] Load: The entire Butyl eluate was loaded onto the SP
column.
[0275] Bed height: All columns were packed with standard 10 cm bed
height.
[0276] Detailed operating procedures carried out with regard to
this two column botulinum toxin complex purification process set
forth in this Example 12 are set forth below.
1. Butyl Hydrophobic Interaction Column
Materials and Reagents Used
[0277] Chromatography System: AKTA purifier 100, GE Healthcare
Resin Type Butyl Sepharose FF, Amersham Pharmacia
Detection: UV (280 nm)
Equilibration Buffer/Wash Buffer #1: 50 mM NaPi, 4 M NaCl, pH
6.0
Wash Buffer # 2: 50 mM NaPi, 2 M NaCl, pH 6.0
Elution Buffer: 50 mM NaPi, 1 M NaCl, pH 6.0
Low Salt Wash Buffer: 50 mM NaPi, pH 6.0
Cleaning Solution: 0.1 N NaOH
Titration Buffer: 500 mM NaPi, pH 7.2
Procedure
Column Packing and Conditioning
[0278] Equilibrate the column with at least 5-10 CV of
Equilibration Buffer or until outlet pH is equivalent to inlet
pH.
Sample Preparation
[0279] Measure the pH of the starting material.
[0280] Add solid NaCl to the clarified culture to the final NaCl
concentration to 4 M.
[0281] Addition of 4M NaCl is an example of how to condition the
clarified culture for use of the clarified culture as a feed liquid
in hydrophobic interaction chromatography.
[0282] Adjust the pH to 5.0 to 6.0 if needed with Titration
Buffer.
Column Loading
[0283] Load the clarified culture (containing 4M NaCl) and collect
the flow through fraction for analysis.
Column Wash # 1 (4 M NaCl Wash)
[0284] Wash the column proteins with 5CV of Equilibration Buffer to
remove impurity.
[0285] Collect the wash fraction for analysis and record the
volume.
Column Wash # 2 (2 M NaCl Wash)
[0286] Wash the column with 15CV of Wash Buffer # 2 to remove
additional impurity proteins. Collect the wash fraction for
analysis and record the volume.
Elution (1 M NaCl Toxin Peak Elution)
[0287] Elute the bound toxin with 5 CV of Elution Buffer. Monitor
the 280 nm absorbance of eluate, begin the collection of eluate
when the 280 nm absorbance starts to increase and stop the
collection of the eluate peak when the 280 nm absorbance reaches
the baseline. Record the volume of toxin elution fraction.
Low Salt Wash (0 M NaCl Impurity Peak Elution)
[0288] Wash the column with 4CV of Low Salt Wash Buffer to remove
residual impurity proteins. Collect the fraction for analysis and
record the volume.
Column Cleaning (0.1 N NaOH)
[0289] Clean the column with 3 CV of Cleaning Solution to
inactivate the residual toxin before the disposal of used
resin.
2. SP Cation Exchange (Post Butyl) Column
Materials and Reagents Used
[0290] Chromatography System: AKTA purifier 100, GE Healthcare
Resin Type: SP Sepharose HP, Amersham Pharmacia
Detection: UV (280 nm)
Dilution, Equilibration and Wash Buffer #1: 20 mM NaCitrate, pH
4.0
Wash Buffer #2: 20 mM NaCitrate, 300 mM NaCl, pH 4.0
Elution Buffer: 20 mM NaCitrate, 400 mM NaCl, pH 4.0
High Salt Buffer: 20 mM NaCitrate, 1 M NaCl, pH 4.0
Cleaning Solution: 0.1 N NaOH
Procedure
Column Packing and Conditioning
[0291] Equilibrate the column with 5-10 CV of Equilibration Buffer
or until outlet pH is equivalent to inlet pH.
Sample Preparation
[0292] Dilute one volume of 1M NaCl Butyl eluate with 4 volume of
Dilution Buffer. Measure the conductivity and pH of the load.
Adjust the pH to 4.0 if needed.
Column Loading
[0293] Apply the above diluted Butyl eluate to SP column and
collect the flow through fraction.
Column Wash # 1 (Equilibration Buffer Wash)
[0294] Wash the SP column with 5CV of Equilibration Buffer.
Continue to collect the eluate as flow through fraction.
Column Wash # 2 (300 mM NaCl Wash)
[0295] Wash the SP column with 4CV of Wash Buffer # 2 to remove
impurity proteins. Record the volume of the wash #2 fraction.
Elution (400 mM NaCl Elution)
[0296] Elute the bound toxin with 3CV of Elution Buffer. Monitor
the 280 nm absorbance of eluate, begin the collection of eluate
when the 280 nm absorbance starts to increase and stop the
collection of the eluate peak when the 280 nm absorbance reaches
the baseline. Record the volume of toxin elution fraction.
High Salt Elution (1 M NaCl)
[0297] Elute the strongly bound impurity proteins with 3CV of High
Salt Buffer. Collect the fraction for analysis and record the
volume.
Column Cleaning
[0298] Clean the SP column with 3 CV of Cleaning Solution to
inactivate the residual toxin before the disposal of used
resin.
Example 13
Robustness of the Two Column APF Chromatography Process for
Purifying a Botulinum Toxin Complex
[0299] The robustness of the two column method of Example 12 was
studied in a series of experiments, as set forth below.
[0300] Culture pH
[0301] The effect of culture pH on toxin purification was
evaluated. A study using cultures grown at pH 5.5 and pH 6.5 as the
starting material for the purification was performed, and it was
found that the recovery from the pH 6.5 culture was slightly lower
than that from the pH 5.5 culture, based on Hc-ELISA results.
[0302] Storage Time
[0303] Toxin was purified from a culture grown at pH 5.5 on the day
of harvesting and after 4-day storage of the culture at 2-8.degree.
C. No difference was found, based on toxin recovery, Butyl and SP
chromatograms, SDS-PAGE, and Hc-ELISA results.
[0304] Column Binding Capacity
[0305] The proposed load on the Butyl column was 12.7 ml culture
per ml resin, or 10 L culture for BPG100 column (with 10 cm bed
height). Butyl and SP columns were tested by loading 4.times. more
culture. SDS-PAGE and Hc-ELISA results indicated little toxin in
the flowthru fractions for both Butyl and SP columns. The capacity
of Butyl and SP column is at least four times greater than that of
the current load. The toxin in SP eluate was pure on SDS-PAGE. The
recovery of Butyl column is 48% and the recovery of SP column was
74%, based on Hc-ELISA. The overall yield is 16 mg toxin per L
culture, based on UV result.
[0306] Process Hold Time
[0307] After harvesting, the culture was processed through Butyl
column on the same day or after overnight storage. Butyl eluate was
normally stored overnight before loading onto the SP column. A
preliminary study showed that the Butyl eluate was stable for up to
4 days, which gave identical chromatogram and SDS-PAGE patterns.
The stability of SP eluate was evaluated by capillary
electrophoresis (CE) and SEC-HPLC. The results showed no difference
among samples stored for up to 2 days. The recovery of toxin after
filtration was also evaluated for these samples. Toxin recovery was
slightly decreased on day 2 compared to day 0, but it was not clear
whether such decrease was due to storage or experimental
variation.
[0308] Cell Density of Culture
[0309] Two times concentrated culture and 2.times. diluted culture
were evaluated by Butyl column chromatography to study the effect
of culture cell density on toxin purification. The chromatograms
from both runs looked identical. The impurity and toxin profile
from both runs were identical on SDS-PAGE. The Hc-ELISA results
(Table 7) showed that the mass balance from both runs were >90%,
while the recovery of 2.times. concentrated culture was
significantly lower than that of 2.times. diluted culture.
Twenty-nine percent toxin was lost before toxin elution for
2.times. concentrated culture, compared with 4% loss for 2.times.
diluted culture.
TABLE-US-00008 TABLE 7 APF toxin mass balance analyzed by Hc-ELISA.
Mass FT 2M 1M 0M Run balance (FlowThrough) Wash Elution Elution
2.times. concent. 91% 11% 18% 53% 9% 2.times. diluted 97% 0% 4% 74%
19%
[0310] Bioburden Studies
[0311] Bioburden was monitored at different steps of the process.
Samples of Butyl load, Butyl eluate after 3 day storage, SP load,
SP eluate, and SP eluate after overnight storage were evaluated.
Some contaminants were noted (.about.<1 CFU/ml to 35 CFU/ml).
The sample with the highest number of contaminants was the Butyl
eluate. Contaminants may be due to the uncontrolled environment in
which purification process was performed.
[0312] Effect of 4M NaCl
[0313] In order to evaluate the effect of 4M NaCl on the toxin in
culture, the culture containing 4M NaCl was kept at 4.degree. C.
overnight and then Butyl and SP column chromatography were
performed. The chromatographic result, SDS-PAGE and Hc ELISA showed
there was no effect of 4M NaCl on the toxin in the culture after
overnight storage.
[0314] Culture Media (3:1:1 Vs 5:1:1)
[0315] Two point five liters of 5:1:1 and 3:1:1 cultures were
processed. The toxin recovery for each of the purifications
analyzed by Hc-ELISA is summarized in Table 8. The toxin purified
from both cultures was pure on SDS-PAGE, which indicates that the
process developed with 3:1:1 culture can be used to purify toxin
from 5:1:1 culture.
TABLE-US-00009 TABLE 8 Toxin recovery based on Hc-ELISA Step 3:1:1
culture 5:1:1 culture Butyl 46% 43% SP 63% 44%
[0316] Working pH for SP Sepharose HP Chromatography
[0317] SP Sepharose HP chromatography was carried out at different
pH values: 3.5, 4.2, and 4.5. It was found that pH 3.5 caused toxin
precipitation in the column, no toxin was eluted with 400 mM NaCl
and very little toxin came out with 1M NaCl. At pH 4.5, toxin did
not bind to the SP column. Preliminary results obtained at pH 4.2
showed that the toxin did not bind as strongly as at pH 4.0 and was
eluted as a broad peak after the wash peak at 300 mM NaCl. The
results indicate that the pH at this step was critical and that the
optimal pH range was narrow.
Example 14
Evaluation of Two Column APF Chromatography Process for Purifying a
Botulinum Toxin Complex
[0318] Various eluents from each of the two columns of the
purification process of Example 12 were evaluated as set forth
below.
[0319] A. Butyl Sepharose FF Chromatography
[0320] Filtered 3:1:1 culture was used as the feed for this
experiment. Before loading the feed (clarified culture obtained
from a Schantz fermentation of a Clostridium botulinum type A [Hall
strain]) onto the Butyl Sepharose FF column (XK50/10, column
diameter 5 cm, bed height 10 cm, column volume: 196 ml), 584.4 g of
NaCl was added to 2500 ml of culture with stirring for .about.30
min. Atypically, the feed pH was adjusted to 5.81 and the running
flow rate was set at 92 cm/h (normal flow rate is 100 cm/h). The
loading volume was 2800 ml.
[0321] After loading, the column was washed with 5CV or 1000 ml of
50 mM NaPi, 4M NaCl, pH 6.0, followed by 15CV or 3000 ml of 50 mM
NaPi, 2M NaCl, pH 6.0. The bound botulinum toxin type A complex was
then eluted from the column with 5CV or 1000 ml of 50 mM NaPi, 1M
NaCl, pH 6.0. After the elution of the botulinum toxin complex, the
strongly bound impurities were washed off the column with 4CV or
800 ml of 50 mM NaPi, pH 6.0. The column was next washed with 2CV
(400 ml) of 0.1N NaOH to inactivate residual toxin before the
disposal of used resin. The chromatogram of the toxin eluent is
shown in FIG. 3.
[0322] FIG. 3 shows that the Butyl column used can provide good
separation of botulinum toxin complex from impurities present with
it in the clarified culture feed liquid. As measured by UV280 nm,
FIG. 3 shows the flow through peak and the peaks of 2M NaCl, 1M
NaCl, 0M NaCl and 0.1N NaOH. Based on the peak size, it was
determined that most impurities were removed in the flow through
fraction. A significant amount of impurities were also removed in
2M NaCl fraction before the elution of toxin in the 1M NaCl
fraction.
[0323] FIG. 3 is a chromatograph obtained from passage of an APF
clarified culture (a 3.1.1 culture) through a Butyl hydrophobic
interaction column. The X axis represents the volume in ml of
liquid (effluent) which has passed through the column. The Y axis
represents the UV absorbance at 280 nm in mAU. In addition, the
conductivity (separate graph line) was monitored during
chromatography.
[0324] As shown by FIG. 3, many protein impurities passed through
the column in about the first approximately 3000 mls. The 4M NaCl
and 2M NaCl wash buffers caused subsequent, though smaller peaks,
showing removal of additional impurities. Use of the 1M NaCl (at
about the 7000 ml volume) caused elution of bound toxin complex
from the column and this was the fraction loaded onto the second
column.
[0325] B. SP Sepharose HP Chromatography
[0326] The axes in FIG. 4 are the same as they are for FIG. 3. The
steps carried out to obtain the FIG. 4 chromatograph were as
follows:
(1) one hundred ml of the Butyl eluate obtained from Example 12
(the Butyl column eluent resulting from FIG. 3) was diluted with
400 ml of 20 mM Na citrate buffer at pH 4.0 (a five times dilution
therefore). The pH of this diluted Butyl eluent was 4.1. (2) four
hundred and sixty-six ml of this feed was then loaded onto the SP
Sepharose HP column (XK26/10, column diameter 2.6 cm, bed height 10
cm, column volume: 53 ml). (3) after loading the column was washed
(at about the volume 450 ml point on the x axis of FIG. 4) with 5CV
or 250 ml of 20 mM Na citrate, pH 4.0. (4) the column was then
washed with 4CV or 200 ml of 20 mM Na citrate, 300 mM NaCl, pH 4.0
(at about the volume 725 ml point on the x axis of FIG. 4). (5) the
column bound botulinum toxin complex toxin was then eluted with 3CV
or 150 ml of 20 mM Na citrate, 400 mM NaCl, pH 4.0 (at about the
volume 925 ml point on the x axis of FIG. 4). (6) after elution of
the column bound toxin complex, the column was further washed with
3CV or 150 ml of 20 mM Na citrate, 1M NaCl, pH 4.0 to elute
strongly bound impurities (at about the volume 1050 ml point on the
x axis of FIG. 4). (7) the column was then cleaned with 3CV or 150
ml of 0.1N NaOH (just after the volume 1200 ml point on the x axis
of FIG. 4).
[0327] The FIG. 4 chromatogram shows elution of a botulinum toxin
type A complex (about 900 kDa molecular weight) just before the
1000 ml volume point on the x axis of FIG. 4.
[0328] FIG. 4 shows that high purified botulinum toxin complex can
be obtained by use of the SP sepharose column subsequent to the
Butyl column. FIG. 3 shows that there was a broad flow through
peak, a small 300 mM NaCl wash peak, 400 mM toxin elution peak and
1M NaCl cleaning peak. As analyzed by SDS-PAGE in FIG. 5B, there
was no visible protein band in flow through fraction, some impurity
protein bands in 300 mM NaCl wash fraction and 1M NaCl cleaning
fraction. Toxin was eluted in 400 mM NaCl elution fraction.
[0329] C. Analytical Results:
[0330] SDS-PAGE: The elution fractions from the Butyl and SP column
chromatography columns were analyzed by SDS-PAGE and the typical
result is shown in FIG. 5A (Butyl column) and FIG. 5B (SP
column).
[0331] FIGS. 5 and 6 are gel electrophoresis records obtained by
use of reduced SDS-PAGE. The left had side of the FIGS. 5 and 6 gel
electrophoresis records is marked vertically with ascending
molecular weights in thousands of Daltons (kDa). The numbers 1 to
6, 1 to 7 or 1 to 8 in FIGS. 5A, 5B and 6 represent the fractions
loaded onto the gels.
[0332] In FIG. 5A: item 1 (gel lane 1) "Mark 12" is the Novex
molecular weight marker of standard molecular masses; lane 2 is the
clarified culture feed liquid; lane 3 is an aliquot from the wash
resulting from use of the flow through ("FT") and 4M wash in the
Butyl column; lane 4 is an aliquot from use of the 2M wash; lane 5
is an aliquot from the tail fraction of the 2M wash; lane 6 is an
aliquot from the fraction of 1M elution; lane 7 is an aliquot from
the tail fraction of 1M elution, and; lane 8 is an aliquot from the
0M wash.
[0333] FIG. 5A shows that the Butyl column removed many impurities
(see lanes 3-5 in FIG. 5A) and provided initially purified
botulinum toxin (see lanes 6-8 in FIG. 5A).
[0334] In FIG. 5B: item 1 (gel lane 1) "Mark 12" is the same
molecular weight marker used in FIG. 5A; lane 2 is the diluted
Butyl column eluent; lane 3 is an aliquot of the column flow
through; lane 4 is an aliquot from the 300 mM wash; lane 5 is an
aliquot from eluant from the column; lane 6 is an aliquot from the
1M wash. FIG. 5B shows that use of an SP column subsequent to use
of a Butyl column provided highly purified botulinum toxin (see
lane 5 in FIG. 5B).
[0335] Hc-ELISA
[0336] Toxin concentration was analyzed by Hc-ELISA, an ELISA assay
to determine the toxin concentration based on the concentration of
toxin heavy chain, and toxin mass balance during the purification
was estimated. Table 9 shows the toxin concentration and step
recovery during Butyl and SP column steps from a typical
purification run. The overall recovery after Butyl and SP was
28.6%.
[0337] SEC-HPLC
[0338] The results from SEC-HPLC showed that the step recovery for
SP chromatography was 42.9%, compared to 62.5% from Hc-ELISA. This
shows that the recovery of botulinum toxin after the SP column step
was approximately 50%.
[0339] Normalized Yield
[0340] The toxin yield was normalized as 22.3 mg (by SEC-HPLC) or
23.4 mg (by Hc-ELISA) per L culture after Butyl chromatography, and
9.6 mg (by SEC-HPLC) or 8.9 mg (by Hc-ELISA) per L culture after SP
chromatography from one run. Thus, using our two column system and
process set forth herein, between about 50 mg to about 90 mg of
botulinum toxin complex can be purified from each 10 L of
fermentation medium clarified culture (as obtained for example from
the Example 6 or Example 7 fermentation processes).
TABLE-US-00010 TABLE 9 Toxin concentration and mass balance in
typical Butyl and SP chromatography steps. Volume Conc Toxin Amt
(ml) (.mu.g/ml) (mg) % Recovery Butyl samples Butyl Load 2800 45.5
127.4 100 Flowthru and Wash 2634 N/A N/A N/A 2M NaCl Wash Peak 336
32.5 10.9 8.6 1M NaCl Elution Peak 404 144.5 58.4 45.8 1M NaCl Post
Elution 443 N/A N/A N/A 0M NaCl Wash 369 19 7.0 5.5 SP Samples SP
Load 466 19 8.9 100.0 Flowthru 708 N/A N/A N/A 300 mM Wash Peak 54
N/A N/A N/A Elution Peak 35 158 5.5 62.5 1M Wash Peak 13 N/A N/A
N/A Cleaning Peak 44 N/A N/A N/A
Example 15
Process for Post Column Chromatography Toxin Complex Stabilization
and Storage
[0341] 1. Development Rationale
[0342] After column chromatography, it is preferred to transfer the
purified botulinum toxin complex into a stable buffer at a desired
concentration by a UF/DF step, followed by sterile filtration to
thereby obtain a toxin suitable for use in a compounding of a
botulinum toxin pharmaceutical composition. The purified botulinum
was stored either in a soluble form in acetate buffer or as an
ammonium sulfate suspension.
[0343] 2. UF/DF Step
[0344] A polyethersulfone Biomax-10 membrane (NMWCO: 10 kDa,
Millipore) was used in the UF/DF step. 50 mM NaAc, pH 4.0 was
chosen as the diafiltration buffer. The SP eluate was ultrafiltered
to .about.1 mg/ml, then diafiltered with 8 diafiltration volumes
(DV) of 50 mM NaAc, pH 4.0.
[0345] Ultrafiltration (UF) is a process for separating extremely
small particles and dissolved molecules from fluids. The primary
basis for the separation is molecular size although secondary
factors such as molecule shape and charge can play a role.
Materials ranging in size from 1,000 to 1,000,000 molecular weight
are retained by ultrafilter membranes, while salts and water pass
through. Colloidal and particulate matter can also be retained.
[0346] Diafiltration (DF) is the fractionation process that washes
smaller molecules through a membrane and leaves larger molecules in
the retentate without ultimately changing concentration. DF can be
used to remove salts or exchange buffers. DF can also remove
ethanol or other small solvents or additives. There are several
ways to perform diafiltration. In continuous diafiltration, the
diafiltration solution (water or buffer) is added to the sample
feed reservoir at the same rate as filtrate is generated. In this
way the volume in the sample reservoir remains constant, but the
small molecules (e.g. salts) that can freely permeate through the
membrane are washed away. Using salt removal as an example, each
additional diafiltration volume (DV) reduces the salt concentration
further. (A diafiltration volume is the volume of sample before the
diafiltration solution is added.) Using 5 diafiltration volumes
will reduce the ionic strength by .about.99% with continuous
diafiltration. In discontinuous diafiltration, the solution is
first diluted and then concentrated back to the starting volume.
This process is then repeated until the required concentration of
small molecules (e.g. salts) remaining in the reservoir is reached.
Each additional diafiltration volume (DV) reduces the salt
concentration further. A diafiltration volume is the volume of
sample before the diluting solution is added. Using 5 diafiltration
volumes will reduce the ionic strength by .about.96% with
discontinuous diafiltration. Continuous diafiltration requires less
filtrate volume to achieve the same degree of salt reduction as
discontinuous diafiltration.
[0347] 3. 0.22 .mu.m Filtration Step
[0348] The low-protein-binding 0.22 .mu.m cellulose acetate (CA)
vacuum bottle-top filter was selected for the filtration step.
[0349] 4. Ammonium Sulfate Precipitation Step
[0350] Ammonium sulfate precipitation was then carried out: 3.5M
ammonium sulfate was added to the 0.22 .mu.m filtered toxin
solution with gentle stirring until the first appearance of
opalescence. The purified bulk toxin was then stored at 2-8.degree.
C.
[0351] 5. Results from a Typical Post-Column Process
[0352] SP eluate was concentrated from 70.5 ml to 18 ml using
Pellicon Biomax-10 (50 cm.sup.2 surface area, Millipore) on a
Labscale TFF system (Millipore) and diafiltered with 8DV of 50 mM
NaAc, pH 4.0. The retentate (post-UF/DF fraction) was collected and
was filtered with Corning 0.22 .mu.m CA filter (Corning 431154).
The UF/DF system was rinsed with acetate buffer. The rinse fraction
was collected. Ten ml of the post 0.22 .mu.m filtrate was stored at
2-8.degree. C. for stability studies. Eight ml of the post 0.22
.mu.m filtrate was subjected to ammonium sulfate precipitation. A
total of 2.8 ml of 3.5 M ammonium sulfate was added into the
filtrate until it became opalescent.
[0353] Toxin recovery was estimated based on UV measurement, which
is shown in Table 10. SDS-PAGE results are shown in FIG. 4.
[0354] In FIG. 6 the lanes shown represent:
Lane 1 is M12, molecular weight standards Lane 2 is SP column
eluate Lane 3 is UF/DF retentate: UF/DF retentate after UF/DF of SP
eluate, diluted to the same amount of loaded protein as Lane 2, for
comparison Lane 4 is UF/DF rinse solution from rinsing UF/DF
membrane after completion of UF/DF membrane Lane 5 is post 0.2
.mu.m filtration; after UF/DF process and after the sample was
filtered with the 0.22 .mu.m filter Lane 6 is post column ammonium
sulphate suspension; after 0.22 .mu.m filter filtration, the sample
was precipitated with ammonium sulphate because the botulinum toxin
complex is stable in ammonium sulphate Lane 7 is UF/DF retentate
(same as lane 3), but undiluted, to show the details
[0355] FIG. 6 tells us that the post column purification process
steps of UF/DF, 0.22 .mu.m filtration, and ammonium sulphate
precipitation do not affect the purity of the botulinum toxin
complex, as determined by SDS-PAGE analysis. Significantly, the
MLD.sub.50 results showed that the potency of the purified bulk
botulinum toxin complex was 2.9-3.7.times.10.sup.7 MLD.sub.50
units/mg.
TABLE-US-00011 TABLE 10 Toxin recovery based on UV measurement
Toxin conc. by UV Vol. Total toxin Recovery Fraction (mg/ml) (ml)
(mg) (%) SP eluate 0.389 70.5 27.4 100 (defined) Post UF/DF 1.260
18.0 22.7 82.8 UF/DF rinse 0.220 14.0 3.1 11.3 Post filtration
1.270 18.0 22.8 83.2 Post AS ppt* N/A (~0.94) ~10.8 N/A N/A *from 8
ml post filtration fraction.
[0356] FIG. 7 is a flowchart of a preferred animal protein free,
two column chromatographic method for purifying a botulinum toxin
type A complex. This is a robust, scalable and cGMP compliant
process for obtaining purified Clostridium botulinum toxin 900 kDa
complex. In FIG. 7 it can be noted that the Butyl eluate is
conditioned for ion exchange chromatography by a five times
dilution with a pH 4 sodium citrate buffer.
[0357] The FIG. 7 process can also be used to obtain pure (i.e. 150
kDa botulinum toxin free of the non-toxin complex proteins) by
loading the SP column eluent onto an ion exchange column in a pH 8
buffer to disassociate the non toxin complex proteins from the 150
kDa botulinum toxin molecule, thereby providing (in the flow
through from the column) a botulinum toxin type A (neurotoxic
component) with an approximately 150 kD molecular weight, and a
specific potency of 1-2.times.10.sup.3 LD.sub.50 U/mg or greater.
This process can also be used to obtain other non toxin components
of botulinum toxin complex (i.e. non toxin hemagluttinin proteins
and/or non toxin non hemaglutinin proteins) by dissociating the
complex into its components and next purifying the dissociated
components.
[0358] The purified toxin complex obtained by our process meets or
exceeds the specifications set forth in Table 1. Additionally, the
typical yield was approximately 100 mg of 900 kDa toxin complex
from a 10 L cell culture, which is higher than the yield obtained
from a Schantz (non-APF) process.
Example 16
Three Column APF Chromatographic Method and System for Purifying a
Botulinum Toxin
Introduction
[0359] We have developed a highly efficient, high yield system and
process for obtaining a botulinum toxin with higher purity and
higher potency than can be obtained using known process or systems.
Our system and process can be used to purify a botulinum toxin
obtained from a fermentation of an organism which can produce a
botulinum toxin, such as a Clostridium bacterium, or of an organism
genetically altered to make a botulinum toxin, such as an E. coli
bacterium or a suitable yeast species inserted with a plasmid which
encodes for botulinum toxin synthesis.
[0360] Preferably, the fermentation of an organism which can
produce a botulinum toxin is an animal product free (APF), or
substantially APF, fermentation because the particular three column
chromatographic process set forth in this Example 16 is optimized
for application to fermentation medium resulting from an APF
fermentation of an organism which can produce, that is release a
botulinum toxin into the APF fermentation medium.
[0361] An APF process or system does not make use of any animal
materials, such as any animal products, animal derived product
(i.e. derived by hydrolysis of an animal protein) or any animal
proteins. The particular process and system in this Example 16 is
an APF process and system for purifying a Clostridium botulinum
type A neurotoxin complex from APF clarified culture.
SUMMARY
[0362] We developed a system and process for purifying a botulinum
toxin from clarified culture. A clarified culture can be obtained
from a research scale or from an industrial scale anaerobic, APF
fermentation of a Clostridium botulinum. Typically a research scale
fermentation results in less than 50 liters of clarified culture
while an industrial scale fermentation results in more than 50 or
more liters (i.e. 100-200 liters) of clarified culture. Analytical
characterization of the botulinum toxin obtained from our Example
16 process and system showed both the purity and potency of the
botulinum toxin obtained is superior to the purity and potency of
botulinum toxins obtained from a Schantz (non-APF) process (i.e. as
set forth in Example 6) or from an alternate chromatographic system
and process for purifying a botulinum toxin (such as the process
and system in Examples 12 to 15. For example, with regard to
purity, the residual nucleic acid content of the botulinum toxin
obtained using our Example 16 process was significantly lower than
(as low as less than 2%) of the residual nucleic acid content of a
botulinum toxin purified according to a Schantz process, such as
the Example 6 process. Additionally, the 50% mouse lethal dose
(MLD50) specific activity (potency) of purified botulinum toxins
obtained using our Example 16 process was significantly higher (up
to 64% higher) that the potency of purified botulinum toxins
obtained from a Schantz process or a modified Schantz process, such
as the Example 6 process.
[0363] FIG. 9 presents is a summary flow chart showing three
processes and systems for obtaining a purified botulinum toxin: the
Example 6 (Schantz, non-APF) process; a preferred embodiment of the
Examples 12-15 APF process (a two column chromatographic process
carried out upon an APF fermentation clarified culture), and; a
preferred embodiment of the Example 16 APF process (a three column
chromatographic process also carried out upon an APF fermentation
clarified culture). The process set forth in this Example 16
differs from the Examples 12-15 processes by, for example, using a
simple centrifugation step as the botulinum toxin is purified. In
FIG. 9 "culture supernatant" for the Example 12-15 Process and for
the Example 16 Process is a clarified APF culture. The "culture"
for the Example 6 process is a non-APF whole culture (not
clarified).
[0364] The Example 16 process and system was optimized for
purifying a Clostridium botulinum type A neurotoxin complex from an
APF clarified culture. Clarified culture can be obtained by
clarifying a Clostridium botulinum APF fermentation medium (i.e.
"culture") using a 0.2 .mu.m depth filter. The clarified culture
was then subjected to ultrafiltration (UF; also called
microfiltration [MF]) and diafiltration (DF) (that is dialyzed)
against 0.1M phosphate buffer at pH 6.0, to concentrate the
clarified culture, followed by centrifugation step and three
orthogonal column chromatography steps.
[0365] We discovered that with our process the botulinum neurotoxin
complex precipitates from the UF/DF retentate, without any need for
acidification to cause the toxin to precipitate. The UF/DF retenate
was centrifuged to separate most of the impurities into a
supernatant. The resulting toxin pellet was resolublized in 0.1M
citrate buffer, pH 4.0 and then further purified using SP Sepharose
HP, Phenyl Sepharose HP and Ceramic Hydroxyapatite type I columns
chromatography to remove residual impurities. We carried out this
APF purification process on 0.5 L, 4 L scale and 10 L clarified
cultures resulting from APF fermentations, and determined that our
process is scalable for use with industrial volume (i.e. 100 L, 200
L) clarified cultures from APF fermentations. The precipitation
steps which are used in our Example 16 process are different from
the Example 6 acid, ethanol, and ammonium sulfate precipitation
steps. Additionally our Example 16 process differs form the
Examples 12-15 APF purification process in that after UF/DF the
high quality toxin is precipitated and most of impurities are
removed in supernatant. The additional chromatography steps in this
Example 16 process as compared to the Example 12-15 process improve
the robustness of the purification process and ensure that
variation during fermentation does not affect the quality of the
purified toxin (botulinum neurotoxin complex).
[0366] Our Example 16 APF process and system for purifying a
botulinum toxin (such as a Clostridium botulinum type A neurotoxin)
from an APF clarified culture using animal product free (APF)
materials can have the following sequential steps upon a botulinum
toxin APF clarified culture: (a) ultrafiltration (UF); (b)
diafiltration (DF); (c) precipitation; (d) extraction, (e) a first
chromatography step; (f) a second chromatography step; (g) a third
chromatography step, and (h) final processing steps so as to obtain
highly purified, biologically active, bulk botulinum toxin suitable
for entering a compounding process for the preparation of a
botulinum toxin pharmaceutical composition. The chromatography
steps can entail use (1) SP Sepharose HP, (2) Phenyl Sepharose SP;
(3) Sepharose HP, (40 Phenyl Sepharose HP, and (5) Ceramic
Hydroxyapatite Type I chromatography columns.
[0367] As shown by FIG. 9, the Example 6 purification process has
three separate precipitation steps, which are acid, ethanol and
ammonium sulfate precipitation steps. Notably the purification
process of this Example 16 does not require use of any of these
three Example 6 purification steps. Elimination of the three
Example 6 precipitation steps results in both a higher yield of
toxin (less toxin lost due to elimination of the three
precipitation steps) and yield of more biologically active toxin
(less toxoid formation due to reduced toxin processing). In the
Examples 12-15 APF chromatography processes UF and DF steps are
carried out only after the chromatographic purifications (see FIG.
9). In this Example 16 process UF and DF steps are carried out both
before and after the chromatographic purifications of the toxin.
Additionally, the Example 16 process follows initial UF and DF
steps with toxin precipitation and extraction steps. Example 16
does not use the same Example 6 precipitation step to purify the
botulinum toxin. Note as shown by FIG. 9, that the Examples 12-15
process makes use of neither initial UF and DF steps, nor any
precipitation and extraction steps prior to chromatography. These
distinctions with regard to the Example 16 process as compared to
the Examples 12-15 processes causes removal of more impurities
(into the supernatant from the filtration steps) and entry of a
higher quality toxin form the clarified culture into the
chromatography steps. The process in Example 16 is different from
the processes in Example 12-15. For example, in the Example 16
process the volume reduction is performed by UF/DF and the
botulinum toxin is almost pure after precipitation. Contrarily, in
the Example 12-15 process the volume reduction is performed by use
of a Butyl column, which also removes a lot of impurities.
[0368] The additional chromatography steps in the Example 16
process result in a robust purification process and ensures that
variation during fermentation will not affect the quality of the
purified neurotoxin complex, that is that our Example 16 process is
a robust process. According to the FDA, the robustness of an
analytical procedure is a measure of its capacity to remain
unaffected by small, but deliberate, variations in control
parameters and provides an indication of its reliability during
normal usage. Generally, our Example 16 process is a robust process
because the Example 16 process can be used to obtain botulinum
toxin of similarly high quality (i.e. with the same high purity and
high potency .+-.0% of either of these two parameters) even though
the botulinum toxin was obtained from different fermentation
cultures of the same Clostridium botulinum. As is well known, since
botulinum toxin is a biologic, the clarified cultures obtained from
use of the same strain (i.e. Hall strain) of the same bacterial
species (i.e. Clostridium botulinum) using the same fermentation
process have natural, inherent variabilities.
[0369] In our Example 16 process, after precipitation a substantial
amount of the impurities had been removed form the extracted toxin.
The Example 16 first chromatography step removed more residual
impurities. The additional two Example 16 chromatography also
remove more impurities.
[0370] Our Example 16 process was used to purify botulinum toxin
from three batches of ten liters of fermentation medium. The
resulting purified botulinum toxin was analyzed using sodium
dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and
size exclusion high performance liquid chromatography (SE-HPLC) and
was found to meet or exceed the Table 1 specifications, showing
therefore that the resulting purified botulinum toxin is suitable
for use for compounding as or in a botulinum toxin pharmaceutical
composition. We obtained a yield of about 20 mg of final purified
botulinum toxin per 10 liters of APF clarified culture.
[0371] In our Example 16 process we can use a cation exchange
chromatography, followed by hydrophobic interaction chromatography,
and thirdly a mixed-mode (i.e. hydroxyapatite) chromatography to
purify botulinum toxin from clarified culture.
[0372] A preferred cation exchange chromatography resin suitable
for use in the first chromatography step of our Example 16 process
can have one or more of the following characteristics: (1) be a
strong cation ("SP") resin; (2) have a total ionic capacity
(mmol/ml resin gel) of from about 0.15 to about 0.20; (3) exclude
from resin capture globular proteins with a molecular weight
greater than about 4 million; (4) comprises a gel matrix which is
about 6% highly cross linked agarose; (5) have a mean bead size of
about 34 .mu.m; (6) have a bead size range of from about 24 .mu.m
to about 44 .mu.m; (7) be usable with a maximum back pressure of
about 0.5 MPa (5 bar, 70 psi); (8) be usable in a chromatography
column at a linear flow rate of up to about 150 cm/hour, at room
temperature using an aqueous buffer. The linear flow rate can be
measured as the volumetric flow rate (cm.sup.3/hr) divided by the
column's cross sectional area in cm.sup.2; (9) have chemical
stability in commonly used buffers as well as in 8M urea, 6M
guanidine hydrochloride, and 70% ethanol, and/or; (10) have pH
stability over the long term in pH 4-13 and have pH stability over
the short term in pH 3-14. Long term pH stability means that resin
is stable over a long period of time upon use of buffers at such
pHs without adverse effects on it's subsequent chromatographic
performance, while short term pH stability means that solutions in
the pH interval given can be used for resin regeneration, cleaning
in place and sanitization without affecting performance of the
resin.
[0373] A preferred cation exchange chromatography resin suitable
for use in the first chromatography step of our Example 16 process
can be a SP Sepharose High Performance resin (available from GE
Healthcare). This resin is a strong cation ("SP") exchange resin
(the functional group is: sulphopropyl group). It is based on
rigid, highly cross-linked, beaded agarose with a mean bead
diameter of 34 .mu.m. It offers high resolution separation
(compared to XL and Fast Flow resins) with a typical flow velocity
of 100 cm/h. It has high physical and chemical stability, very high
reproducibility and is useful for low pressure, production scale
chromatography.
[0374] A preferred hydrophobic interaction chromatography resin
suitable for use in the second chromatography step of our Example
16 process can have one or more of the following characteristics:
(1) degree of substitution: 25 .mu.mol phenyl group/ml medium; (2)
the medium matrix is 6% highly cross-linked agarose; (3) the mean
bead size is 34 .mu.m; (4) the exclusion limit is 4,000 kDa; (5)
the maximum back pressure is 0.5 MPa (5 bar, 73 psi); (6) useable
at a linear flow rate up to 150 cm/h; (7) have pH stability in as
working range of pH 3-13, and for cleaning in place between pH:
2-14, and/or (8) be autoclavable at 120 deg C., pH 7 for 30
min.
[0375] A preferred hydrophobic interaction chromatography resin
suitable for use in the second chromatography step of our Example
16 process can be a Phenyl Sepharose High Performance resin
(available form GE Healthcare). This resin is a strong hydrophobic
interaction resin (the functional group is: phenyl group). It is
based on rigid, highly cross-linked, beaded agarose with a mean
bead diameter of 34 .mu.m. It offers high resolution separation
(compared to XL and Fast Flow resins) with a typical flow velocity
of 100 cm/h. It has high physical and chemical stability, very high
reproducibility, and is useful for low pressure production scale
chromatography.
[0376] A preferred mixed-mode chromatography resin suitable for use
in the third chromatography step of our Example 16 process can have
one or more of the following characteristics: (1) pH stability at
pH 5.5 to 14; (2) can be regenerated in trisodium phosphate; (3) is
usable at a flow rate up to a maximum flow velocity of about 5,000
cml/hour; (4) have a dynamic binding capacity (lysozyme) of >25
mg per gram resin; (5) have a nominal particle size of 40 .mu.m;
(6) have a nominal pore size of between about 500 .ANG. and about
1,000 .ANG..
[0377] A preferred mixed mode chromatography resin suitable for use
in the third chromatography step of our Example 16 process can be a
ceramic hydroxyapatite type I (40 .mu.m) (available from Bio-Rad)
as a CHT.TM. ceramic hydroxyapatite resin), such as a spherical
macroporous form of hydroxyapatite. The binding mechanism of such
resins is based on a mixed-mode interaction with Ca.sup.2+ as the
positively-charged functional group and the PO.sub.4.sup.3- as the
negatively-charged functional group. It has the separation
properties of crystalline hydroxyapatite, but can be used
reproducibly for several hundred cycles at high flow rates and in
large columns. CHT ceramic hydroxyapatite, Type I, differs from the
crystalline form in its high protein binding capacity and higher
capacity for acidic proteins. Type I has been sintered at different
times and lower temperatures than Type II, resulting in differing
dynamic capacities; the dynamic capacity of Type I is higher and
Type I has a 40 .mu.m particle size.
[0378] Purification Process
[0379] Our Example 16 process and system is an APF chromatographic
process for purifying the botulinum toxin complex present in the
clarified culture obtained from an APF culture and fermentation of
a Clostridium bacterium. Thus, the starting material used in this
Example 16 process was clarified culture. The clarified culture was
obtained by filtering the whole culture obtained from an APF
botulinum toxin culture and fermentation process (such as set forth
in the initial parts of Example 6) with depth filter, followed by
0.2 .mu.m filtration.
[0380] I. Day 1: UF/DF and Pellet Wash (Steps 1-6)
[0381] (1) The clarified culture was ultrafiltered (UF) ten to
fifteen times with a Millipore 300K Biomax membrane; (2) the
ultrafiltered clarified culture was diafiltered (DF) with 0.1M NaPi
at pH 6.0 until the permeate absorption was less than 0.3 at 280
nm; (3) the UF/DF retenate was removed and the filtration membranes
rinsed twice with 0.1M NaPi pH 6.0; (4) the UF/DF retentate and the
twice rinsed solutions were combined to provide a final UF/DF
retentate; (5) the final UF/DF retentate was centrifuged at 3000
rpm for 20 min at room temperature and the supernatant removed
(decanted); (6) the resulting pellets were rinsed (to remove
residual supernatant and therefore more impurities) with 0.1M Pi,
pH 6.0, and again centrifuged followed by removal of supernatant
(the supernatant contained most of the many impurities) and storage
of the resulting pellet at 2-8.degree. C. for less than 96
hours.
[0382] In step (6) 0.1M Pi pH 6 was the preferred pellet rinse
buffer. The effect of rinsing buffer on impurity removal was
evaluated by rinsing pellets with 0.1M Pi buffer at pH 4, 5 and 6
and the rinsed solutions were analyzed by SDS-PAGE. The results
indicated that 0.1M Pi at pH 6 removed the most impurities in that
rinse step.
[0383] The UF/DF membrane used can be a Millipore Biomax or a
Regenerated cellulose membrane filters with either 100K, 300K or
500K MWCO. We analyzed the filtrate and retentate by SDS-PAGE and
Western Blotting. A Biomax-300 membrane was the preferred membrane
for the UF/DF of clarified culture based on the results of
contaminant removal, toxin recovery and flow rate. The DF buffer
used in the UF/DF process was evaluated by diafiltering the
clarified culture with either 1M NaCl in 0.1M Pi pH 6.0, 4M urea in
0.1M Pi pH 6.0, 0.2% octyl glucoside in 0.1M Pi pH 6.0, or 0.1M Pi
pH 6.0. The filtrate and retentate were analyzed by SDS-PAGE and
Western Blotting. The results indicated that use of 1M NaCl
enhanced the removal of impurity proteins in the DF process. The
0.1M Pi pH 6.0 was selected as a preferred DF buffer. To prevent
toxin spill during UF/DF, the inlet pressure of UF/DF was
maintained at approximately 10 psi by adjusting the pump speed,
while outlet valve was open.
[0384] As set forth above, the UF/DF retentate of clarified culture
was used in this experiment. To determine retenate relative
purities each of 2 ml retentate samples were mixed with either 0,
0.1, 0.3, 0.5, 1, 1.5, or 2 ml of 14% PEG 8000 solution. The
mixtures were kept at 4.degree. C. overnight and then clarified by
centrifugation. The supernatants and pellets were analyzed by
SDS-PAGE. The results (FIG. 10) indicate that toxins were in the
pellets and the impurity proteins were in the supernatants for all
samples tested, including 0 ml PEG control sample.
[0385] Surprisingly, the results showed that after DF, the toxin in
UF/DF retentate was precipitated without any precipitant being
added. Lane 6 in FIG. 10 shows a typical result for toxin purified
from UF/DF with the unanticipated precipitation of toxin
occurring.
[0386] The effect of DF volumes on toxin precipitation was
evaluated by collecting retentate samples at various diafiltration
volumes (DV). The collected retentates were centrifuged and the
pellets were washed with buffer. The supernatant and pellets from
different DVs were evaluated by SDS-PAGE. The results indicate that
toxin began precipitating from UF/DF retentate when the DVs were
higher than three. To ensure the removal of small molecular weight
impurities and the precipitation of toxin, more than seven DVs or
the permeate absorbance at 280 nm<0.3 were selected in DF.
[0387] The effect of DF buffer on toxin precipitation were
evaluated by dialyzing retentates in either 0.1M Pi pH 6, 0.1M
citrate pH 6 or fermentation media overnight at 4.degree. C. After
dialysis, the retentates were centrifuged. The toxin in pellets was
extracted with 0.1M citrate, pH 4. The supernatant and pellet
extract were analyzed by toxin heavy chain enzyme linked
immunosorbent assay (Hc ELISA) and SDS-PAGE. The results indicate
that the toxin in retentate was precipitated when the dialysis
buffer was 0.1M Pi pH 6 and 0.1M citrate pH 6, but not fermentation
media. This result indicates that the toxin precipitation from
retentate was due to the removal of small molecules during the DF
or dialysis, not due to physical shear in UF/DF, since the toxin
was precipitated after dialysis.
[0388] In FIG. 10, the following materials were loaded onto the
lanes indicated:
Lane 1: an M12 molecular weight standard Lane 2: supernatant from
the UF/DF retenate Lane 3: a sample of the bulk toxin resulting
from the Example 6 process Lane 4: Supernatant from the first wash
of precipitate Lane 5: Supernatant from the second wash of
precipitate Lane 6: a sample of the toxin purified from the UF/DF
and precipitation process of this Example 16 Lane 7: a sample of
the bulk toxin resulting from the Example 6 Lane 8: a M12 molecular
weight standard Lane 9: a pre-stained molecular weight marker
[0389] The toxin purified by steps (1) to (6) (UF/DF and
precipitation) based on visual inspection of SDS-PAGE gel still
contained a small amount of impurities, including protein and
nucleic acid, although SDS-PAGE clearly showed that by this stage
of the process most impurities had already been removed. To further
purify the botulinum toxin so as to render it useful for
compounding in a pharmaceutical composition the following column
chromatography process was used.
[0390] II. Day 2: Extraction and SP Column (Steps 7-13)
[0391] (7) toxin was extracted from the pellets with 0.1M citrate
buffer, pH 4.0 with the volume equal to 5% to 10% of the clarified
culture volume; (8) the extracted toxin solution was centrifuged at
8000 rpm for 20 minutes at room temperature; (9) the extracted
toxin was diluted with an equal volume of water for injection (WFI)
and loaded onto an SP column.
[0392] In step (7) 0.1M citrate at pH 4.0 was the preferred toxin
extraction buffer. The effect of extraction buffer on toxin
extraction was evaluated by comparing the five potential extraction
buffers: 0.1M Pi pH 6.0; 0.1M citrate pH 6.0; 0.05M citrate plus
0.1M acetate pH 6.0; 0.2M acetate pH 4, and; 0.1M citrate pH 4.0,
in a side by side experiment. The extracted toxins were analyzed by
SDS-PAGE. The result indicated that 0.1M citrate pH 4 was most
effective in extracting toxin from the pellets.
[0393] (10) the SP column was washed (to remove impurities) with
170 mM NaCl, 50 mM citrate buffer, pH 4.0; (11) the toxin was
eluted from the SP column with 400 mM NaCl, 50 mM citrate buffer,
pH 4.0; (12) the SP column eluate was mixed with an equal volume of
4M NaCl, 50 mM citrate, pH 6.0; (13) the diluted toxin eluate was
filtered with a 0.2 .mu.m filter and kept at 2-8.degree. C.
overnight.
[0394] We evaluated Macroprep High S; SP Sepharose Fast Flow; SP
Sepharose High Performance; Source 30S; Source 15S, and Poros HS20
chromatography resins and determined that the Source 15S resins
provided good separation between toxin and impurities. The binding
capacity of Source 15S resin was approximately 7-10 mg toxin per ml
at the flow rate of 100 cm/h.
[0395] A SP Sepharose HP resin was selected because it provided a
good separation between the botulinum toxin and impurities and
because the bead size of this resin is suitable for large scale,
low pressure chromatography. We found that with a SP Sepharose HP
at pH>5.0 and conductivity>21 mS/cm, the toxin did not bind
to the SP Sepharose HP column resin. We developed preferred
conditions for toxin purification with a SP Sepharose HP column
which include use of: 50 mM citrate buffer at pH 4.0; a resin bed
height of 10 cm.+-.1 cm; use of as linear flow rate of 100 cm/hour;
use of 1:1 dilution of toxin sample with water for loading; elution
of impurities from the column using 150-170 mM NaCl, 50 mM citrate
pH 4.0, and; elution of bound botulinum toxin from the column resin
using 400 mM NaCl, 50 mM citrate pH 4.0.
[0396] The toxin eluted from SP column contains a high salt level.
Initially, the eluate from the SP Sepharose HP column was mixed at
1:1 ratio with 1.5M ammonium sulfate (AS), 50 mM sodium phosphate,
pH 6.0 for loading onto the phenyl column. It was observed that the
toxin was not stable in this buffer and tended to form white
aggregates. The replacement of phosphate buffer with citrate buffer
pH 6.0 prevented toxin aggregation. After loading onto the SP
column, the bound toxin was initially eluted with either 0.3 GE
Healthcare M AS or 0M AS. At 0.3M AS, toxin was eluted in a broad
peak with a long tail, while at 0M AS, toxin was eluted in a sharp
peak. The SP column chromatography conditions were further
optimized by using NaCl to replace AS. The eluate from SP Sepharose
HP was mixed at 1:1 ratio with 50 mM sodium citrate, 4M NaCl, pH
6.0. The diluted botulinum toxin eluate was filtered with a 0.2
.mu.m filter and kept at 2.degree. C. to 8.degree. C.
overnight.
[0397] II. Day 3: Phenyl and HA I Columns (Steps 14-19)
[0398] (14) the filtered and diluted toxin was added to a Phenyl
column; (15) the column was washed with 2M NaCl, 50 mM Pi, pH 6.0,
followed with 1.0M NaCl, 50 mM Pi, pH 6.0; (16) the toxin was
eluted from the Phenyl column with 0M NaCl, 50 mM Pi, pH 6.0; (17)
the Phenyl eluate was mixed with water and loaded onto a HA I
column; (18) the HA I column was washed with the following buffers
sequentially:
[0399] (a) 20 mM Pi, pH 6.0
[0400] (b) 500 mM NaCl, 20 mM Pi, pH 6.0
[0401] (c) 20 mM Pi, pH 6.0
[0402] (d) 100 mM Pi, pH 6.0;
[0403] (19) the toxin was eluted from the HA I column with 300 mM
Pi, pH 6.0; (20) the eluted toxin was filtered with a 0.2 .mu.m
filter and kept at 2-8.degree. C. overnight.
[0404] After loading onto the Phenyl, the Phenyl column was washed
with 50 mM NaPi, 1M NaCl, pH 6.0, and the toxin was eluted from the
Phenyl with 50 mM NaPi, pH 6.0. The purified toxin from Phenyl
Sepharose HP column had a purity of 98% with monodispersity, based
on SEC-HPLC and light scattering analysis.
[0405] The botulinum toxin eluate from the Phenyl Sepharose HP
column was diluted 10 times with water prior to being and loaded
onto the Ceramic Hydroxyapatite Type I (HA I) column (bed height 10
cm), which was equilibrated with 10 mM sodium phosphate, pH 6.0.
After loading, the HA I column was washed with 100 mM, 300 mM and
500 mM NaCl in 10 mM sodium phosphate pH 6.0, followed with 100 mM,
300 mM and 500 mM Pi pH 6.0. Based on UV 280 nm, toxin was eluted
at 300 mM sodium phosphate in one peak.
[0406] We carried out an experiment which determined that anion
exchange column chromatography (such as Q Sepharose and DEAE
Sepharose resins) should not be used in toxin column chromatography
purification because such columns can cause the toxin band pattern
to change. Briefly, the experiment was carried out by diluting
Phenyl column eluate 10 times with water and loaded onto a Q column
(a first Q column), equilibrated with 10 mM Pi, pH 6. After
loading, the column was eluted with the step gradient of 100 mM,
150 mM, 200 mM, 300 mM and 500 mM NaCl. The fractions were
collected and analyzed by SDS-PAGE. The fractions from the 150
mM-200 mM NaCl elution was diluted with water and reloaded to Q
Sepharose column (a second Q column). The column was eluted by
using the same step gradients as the first Q column.
[0407] IV. Day 4: Final UF/DF and Ammonium Sulfate Precipitation
(Steps 21-24)
[0408] (21) the HA column was ultrafiltered to the concentration of
1.5 mg botulinum toxin/ml; (22) the concentrated toxin was
diafiltered against 50 mM Pi, pH 6.0 for 4 DV; (23) the resulting
UF/DF toxin was filtered with a 0.2 .mu.m filter; (24) 3.5M
ammonium sulphate (AS) was slowly added to the filtered toxin to
the concentration of 1.2M AS.
[0409] Analytical Results
[0410] The results of this Experiment 16 (steps 1-24) can be
summarized as follows:
[0411] 1. Potency. The MLD50 of final purified botulinum toxin
obtained applying the Example 16 process to different batches of
APF clarified culture was determined to be between
6.25.times.10.sup.7 units/mg and 9.70.times.10.sup.7 units/mg.
[0412] Noting that the MLD 50 of a botulinum toxin obtained from
the Example 6 process was between 2.4 to 5.9.times.10.sup.7
units/mg, we therefore obtained by use of our Example 16 process a
toxin with a potency up to 64% higher (9.70.times.10.sup.7
units/mg) than the highest potency (5.9.times.10.sup.7 units/mg)
typically obtained or required of a purified botulinum toxin
suitable for compounding into a botulinum toxin pharmaceutical
composition, and as obtained from a Schantz process.
[0413] 2. Purity. Residual nucleic acids (dsDNA) remaining in the
final purified botulinum toxin (dsDNA) obtained applying the
Example 16 process to different batches of APF clarified culture
was determined to be between 31 and 76 ng dsDNA per mg of the
purified botulinum toxin. Noting that an Example 6 dsDNA was
between 684 and 1682 ng dsDNA/mg purified botulinum toxin, the
Example 16 process can therefore provide highly purified botulinum
toxin with less than 2% of the amount of residual nucleic acids
present after a Schantz process botulinum toxin purification.
[0414] Botulinum toxin purified using our Example 16 system and
process also had an optical density as measured by absorbance of
light at 260 nm and 278 nm (OD A260/278) between 0.50 and 0.56. Our
Example 16 process can generate more than 100 mg of highly pure and
potent botulinum toxin from 50 liters of APF clarified culture.
[0415] In our Example 16 process, the UF/DF step can take only
about 2-3 hours. Additionally, the botulinum toxin in the UF/DF
retentate was precipitated without adding acid, ethanol or AS,
contrary to the Schantz process. After precipitation, the extracted
toxin was visually pure on SDS-PAGE. The first SP column removes
residual impurities. The additional Phenyl and HA I columns were
used to improve the robustness and ruggedness of the purification
process and ensured that variation during fermentation did not
affect the quality of the purified neurotoxin complex.
[0416] In the Examples 12-15 process, the toxin was purified by a
simple two columns chromatography process. The NaCl was added to
clarified culture to reach 4.0M NaCl before applying to Butyl
column. The Butyl column reduced the process volume, removed a lot
of impurities and the botulinum toxin purity after Butyl column was
approximately 40%. The toxin sample from the Butyl column was
adjusted to pH 4.0 before applying to SP column. After SP column,
the botulinum toxin obtained was pure.
[0417] In the Schantz (i.e. Example 6) process, the starting
material is a non-APF whole culture. The toxin and cell debris are
precipitated by acid precipitation, followed by 0.1 .mu.m
microfiltration. The nucleic acid is then digested by using DNase
and RNase, followed by UF/DF using 100K MWCO membrane. The UF/DF
step removes the low molecular weight impurities. The toxin is
further purified by acid, ethanol and AS precipitations.
[0418] As shown by FIG. 9, the starting materials for both the
Examples 12-15 and Example 16 process is clarified culture. The
final step for both the Examples 12-15 and Example 16 process is
UF/DF, 0.2 .mu.m filtration and AS precipitation. In the Examples
12-15 process the first Butyl column step is used to reduce process
volume, whereas in our Examples 16 process it is the UF/DF step
which serves to reduce the culture volume. Additionally, in the
Examples 12-15 process impurities are removed by the two column
chromatography steps, whereas in our Example 16 process most of
impurities are removed by UF/DF and precipitation step.
[0419] Some of the differences between the Example 16 process and
the Schantz process are summarized in Table 12.
TABLE-US-00012 TABLE 12 Process differences between Example 16
Process and the Schantz (Example 6) Process Example 16 Process
Schantz Process Starting material Clarified APF culture Non-APF
whole culture UF/DF Perform in the first Perform after
acidification, step; use Biomax 300 microfiltration, nucleic acid
filter. digestion and extraction Concentration UF/DF. Volume
adjustment. adjustment in final processing Process time Four days.
About ten days. Purification UF/DF and Acidification, method
precipitation; microfiltration, nucleic acid column chromatography.
digestion and extraction, UF/DF; acid, ethanol and AS
precipitations.
[0420] As shown by Table 12 important differences are starting
material and purification method. The starting material of the
Example 16 process is clarified APF culture, while the Schantz
process uses non-APF whole culture. Additionally, in our Example 16
process, the botulinum toxin is purified by UF/DF and
precipitation; and column chromatography steps whereas in the
Schantz process, the botulinum toxin is purified by acidification,
microfiltration, nucleic acid digestion and extraction, UF/DF, acid
precipitation, ethanol precipitation and AS precipitation.
[0421] In addition to the process differences listed in Table 12,
the botulinum toxin purified using our Example 16 process results
in higher quality (higher purity and potency) botulinum toxin as
compared to the botulinum toxin obtained using the Example 6
Schantz process.
ADVANTAGES OF OUR INVENTION INCLUDE
[0422] 1. No component or substance derived from animal source is
used in the process. Specifically, use of DNase and RNase are
eliminated. 2. Between about 20 mg to about 50 mg of high quality
botulinum toxin type A complex with the characteristics set forth
in Table 1 can be obtained per 10 liters of fermentation medium. 3.
The purified toxin is obtained from a process which is robust,
scalable, validatable, and cGMP compliant. Robust means the process
is reproducibility even upon an about +10% change in one or more of
the process parameters. Validatable means the process consistently
yield purified toxin with the table I characteristics. cGMP means
that the process can be easily converted to a manufacturing process
that complies with FDA required current Good Manufacturing
Practices. 4. The potency of the final purified botulinum toxin
complex meets or exceeds the potency (as determined by the MLD50
assay) of purified botulinum toxin complex obtained from a Schantz
or modified Schantz process. 5. Elimination of any precipitation
steps to purify a botulinum toxin complex or use of a precipitation
step only after a UF/DF step.
[0423] Various publications, patents and/or references have been
cited herein, the contents of which, in their entireties, are
incorporated herein by reference.
[0424] Although the present invention has been described in detail
with regard to certain preferred methods, other embodiments,
versions, and modifications within the scope of the present
invention are possible. For example, a wide variety of animal
product free systems and processes (including chromatographic
botulinum toxin purification processes) are within the scope of the
present invention.
[0425] Accordingly, the spirit and scope of the following claims
should not be limited to the descriptions of the preferred
embodiments set forth above.
* * * * *