U.S. patent application number 10/703866 was filed with the patent office on 2004-05-20 for stabilizing formulation for ngf.
Invention is credited to DeYoung, Linda R., Lam, Xanthe M., Nguyen, Tue H., Powell, Michael F..
Application Number | 20040097417 10/703866 |
Document ID | / |
Family ID | 32303363 |
Filed Date | 2004-05-20 |
United States Patent
Application |
20040097417 |
Kind Code |
A1 |
DeYoung, Linda R. ; et
al. |
May 20, 2004 |
Stabilizing formulation for NGF
Abstract
Formulations are provided comprising NGF and acetate-containing
buffer from pH 5 to 6 that provide enhanced stability of NGF for
use in promoting nerve cell growth, repair, survival,
differentiation, maturation or function.
Inventors: |
DeYoung, Linda R.; (El
Granada, CA) ; Lam, Xanthe M.; (Daly City, CA)
; Nguyen, Tue H.; (San Mateo, CA) ; Powell,
Michael F.; (San Francisco, CA) |
Correspondence
Address: |
HELLER EHRMAN WHITE & MCAULIFFE LLP
275 MIDDLEFIELD ROAD
MENLO PARK
CA
94025-3506
US
|
Family ID: |
32303363 |
Appl. No.: |
10/703866 |
Filed: |
November 6, 2003 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10703866 |
Nov 6, 2003 |
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09619059 |
Jul 18, 2000 |
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09619059 |
Jul 18, 2000 |
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08746073 |
Nov 6, 1996 |
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6090781 |
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60046874 |
Nov 7, 1995 |
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Current U.S.
Class: |
514/8.4 |
Current CPC
Class: |
A61K 38/185
20130101 |
Class at
Publication: |
514/012 |
International
Class: |
A61K 038/18 |
Claims
What is claimed is:
1. A pharmaceutical composition, comprising a pharmaceutically
effective amount of nerve growth factor and a pharmaceutically
acceptable acetate-containing buffer.
Description
[0001] This application is a continuation of U.S. patent
application Ser. No. 08/746,073, filed on Nov. 6, 1996, now U.S.
Pat. No. 6,090,781, which claims priority to U.S. Provisional
Patent Application No. 60/046,874, having an effective filing date
of Nov. 7, 1995, as properly and timely obtained by the petition
dated Nov. 5, 1996, under 37 CFR .sctn.1.53(b)(2)(ii) from U.S.
patent application Ser. No. 08/554,685, filed Nov. 7, 1995, the
contents of which are incorporated herein by reference.
BACKGROUND
[0002] 1. Field of the Invention
[0003] This invention relates to formulations of nerve growth
factor ("NGF") and their use to induce nerve cell growth,
differentiation, survival, repair, maturation, or function in vivo
or ex vivo. More particularly, this invention relates to such
pharmaceutical compositions having increased stability and
solubility characteristics for the NGF component, particularly
human recombinant NGF ("rhNGF"), and those making possible the
ability to create stable forms thereof for safe, effective
therapeutic administration to human subjects.
[0004] 2. Description of Related Disclosures
[0005] Nerve growth factor (NGF) is a neurotrophic factor required
for the growth and survival of sympathetic and sensory neurons
during development and in mature animals (1). Clinical indications
for recombinant human NGF include peripheral sensory neuropathy and
Alzheimer's disease. For example, the systemic administration of
NGF has been shown to reduce the sensory neuropathy induced by
administration of cisplatin and taxol to mice (2,3). In recent
clinical trials, NGF has been administered to humans to improve
sensory function in diabetic neuropathies (4).
[0006] NGF is currently being developed as a liquid parenteral
formulation. The protein stability is complicated beyond the usual
chemical and physical degradation pathways due to the dimeric
structure of NGF. Protein stability can be further complicated when
recombinant protein is a mixture of C-terminally clipped NGF
variants. The crystal structure of murine NGF shows 3 antiparallel
pairs of b-strands forming a flat surface through which the
monomers dimerize (5); the dimer dissociation constant is
.ltoreq.10.sup.-13 M (6, 7). The rearrangement of monomers within
dimers, towards an equilibrium dimer distribution, complicates
quantification of NGF dimer degradation.
[0007] There exists a need for formulations containing NGF that
lead to NGF stability while being safe and effective for
therapeutic administration to mammals, particularly human
subjects.
SUMMARY
[0008] The present invention is based on the finding of formulation
conditions and methods for stability of NGF in a liquid
formulation. It is an object of the present invention to provide a
suitable formulation of NGF with enhanced stability of NGF to
provide effective induction of nerve cell growth, survival,
differentiation, maturation, repair, or function, preferably in
vivo or ex vivo. In various embodiments the formulations can have
enhanced stability to agitation, freezing, thawing, light, or
storage. It is another object of the invention to provide a stable
NGF formulation for use in treating a mammal, preferably human, in
need of NGF treatment so as to provide a therapeutically effective
amount of NGF. It is further object to provide an NGF formulation
with enhanced consistency for improved application to the neuron or
mammal. These and other objects will become apparent to those
skilled in the art.
[0009] The above objects are achieved by providing an NGF
formulation comprising an effective amount of NGF in a
pharmaceutically acceptable acetate buffer, preferably sodium
acetate. In a specific embodiment this formulation contains about
0.1 to 2.0 mg/ml NGF in an acetate buffer from 5 to 50 mM, from pH
5 to 6. The formulation can optionally contain a pharmaceutically
acceptable diluent, a pharmaceutically acceptable salt, preferably
sodium chloride, or a preservative, preferably benzyl alcohol.
[0010] In another embodiment the invention provides a method of
producing an NGF formulation produced by the steps including
formulating NGF and acetate, and optionally sodium chloride, and
further optionally a preservative.
[0011] In another embodiment a method is presented by which NGF
dimer degradation is quantitated independent of dimer exchange.
BRIEF DESCRIPTION OF THE DRAWINGS
[0012] FIG. 1 depicts the dependence of NGF aggregate formation at
37.degree. C. on formulation buffer and pH, quantitated by
size-exclusion chromatography, (.diamond.) succinate pH 4.2;
(.DELTA.) succinate pH 5.0; (.quadrature.) succinate pH 5.8; (X)
succinate pH 5.0 with 0.05% Tween 20; (.tangle-solidup.) acetate pH
5.0; and (.box-solid.) acetate pH 5.8.
[0013] FIG. 2 depicts representative RP-HPLC chromatograms for NGF
in succinate buffer at pH 5.0 (a) -70.degree. C. control and (b)
after 38 days of incubation at 37 degrees C.
[0014] FIG. 3 depicts semilogarithmic plot of the percent NGF
monomer remaining after incubation at 37.degree. C. for various
lengths of time as quantitated by RP-HPLC, (.diamond.) succinate pH
4.2; (.DELTA.) succinate pH 5.0; (.quadrature.) succinate pH 5.8;
(X) succinate pH 5.0 with 0.05% Tween 20; (.tangle-solidup.)
acetate pH 5.0; and (.box-solid.)acetate pH 5.8. Curves are first
order fits to the data.
[0015] FIG. 4 depicts representative IEC chromatograms for NGF in
acetate buffer at pH 5.0 after 38 days of incubation at (solid
line) -70.degree. C. and (dashed line) 37.degree. C. Each dimer
appears as a triplet in the chromatogram due to N-terminal Ser to
Gly (S1G) conversion (13). The earliest peak in the triplet is the
parent dimer, followed by a dimer with a single Ser to Gly
conversion, and finally a dimer with a Ser to Gly conversion in
both chains.
[0016] FIG. 5 depicts time dependence of the loss of NGF 118/118
and 117/120 dimers, by IEC, on incubation at 37.degree. C.,
(.DELTA.) succinate pH 5.0; (.quadrature.) succinate pH 5.8; (X)
succinate pH 5.0 with 0.05% Tween 20; (.tangle-solidup.) acetate pH
5.0; and (.box-solid.) acetate pH 5.8.
[0017] FIG. 6 depicts RP-HPLC chromatograms showing the stability
of NGF after 1.6 years at
[0018] (dashed line) 5.degree. C. and (solid line) -70.degree. C.
The major degradation product at 5.degree. C. is Asn93 to iso-Asp93
conversion.
[0019] FIGS. 7A and 7B depict comparisons of NGF (solid line)
-70.degree. C. control and (dashed line) 5.degree. C. IEC
chromatograms after 1.6 years of incubation in acetate buffer at pH
5.0, (FIG. 7A) no acid treatment, and (FIG. 7B) acid treatment of
samples prior to analysis.
[0020] FIG. 8 depicts RP-HPLC chromatograms of 0.1 mg/ml rhNGF in
10 mM acetate at pH 5.5 and 142 mM NaCl stored at 5.degree. C.
(solid line), 25.degree. C. (dashed line), and 40.degree. C.
(dotted line) for 3 months. Peak (a) contains di-oxidized rhNGF;
peak (b) contains deamidated rhNGF; peak (c) contains mono-oxidized
rhNGF; peak (d) contains Iso-aspartate; peak (e) contains 120
rhNGF; peak (f) contains 118 rhNGF; peak (g) contains N-terminally
clipped rhNGF; peak (h) contains misfolded rhNGF; and peak (i)
contains protein eluted at gradient ramp.
[0021] FIG. 9 depicts determination of rhNGF monomers (118 and 120)
remaining in rhNGF formulations after 12 months at 5 degrees C. by
reversed-phase HPLC. Formulation A (-.crclbar.-) contains 2 mg/ml
rhNGF (142 mM NaCl, 10 mM acetate, pH 5.5); formulation B
(-.quadrature.-) contains 0.1 mg/mL rhNGF (136 mM NaCl, 20 mM
acetate, pH 5.5); formulation C (--.diamond.--) contains
formulation B plus 0.9% BA; formulation D (--x--) contains
formulation B plus 0.25% phenol; formulation E (---+---) contains
0.1 mg/mL rhNGF (136 mM NaCl, 20 mM acetate, 0.01% F68, pH 5.5);
formulation F (-.DELTA.--) contains formulation E plus 0.9% BA; and
formulation G (--.box-solid.--) contains formulation E plus 0.25%
phenol.
[0022] FIG. 10 depicts determination of rhNGF monomers (118 and
120) remaining in rhNGF formulations after 9 months at 25 degrees
C. by reversed-phase --HPLC. Formulation A (-.crclbar.-) contains 2
mg/ml (10 mM acetate, pH 5.5); formulation. B (-.quadrature.-)
contains 0.1 mg/ml (20 mM acetate, pH 5.5); formulation C
(--.diamond.--) contains formulation B plus 0.9% BA; formulation D
(--x--) contains formulation B plus 0.25% phenol; formulation E
(--+--) contains 0.1 mg/mL (20 mM acetate, 0.01% F68, pH 5.5);
formulation F (-.DELTA.--) contains formulation E plus 0.9% BA; and
formulation G (--.circle-solid.--) contains formulation E plus
0.25% phenol.
[0023] FIG. 11 depicts effect of preservative on Iso-aspartate
formation of rhNGF in liquid multi-dose formulations stored at 5
degrees C. for 12 months as determined by RP-HPLC. Formulation A
(-.crclbar.-) contains 2 mg/mL (10 mM acetate, pH 5.5); formulation
B (-.quadrature.-) contains 0.1 mg/mL (20 mM acetate, pH 5.5);
formulation C (--.diamond.--) contains formulation B plus 0.9% BA;
formulation D (--x--) contains formulation B plus 0.25% phenol;
formulation E (--+--) contains 0.1 mg/mL (20 mM acetate, 0.01% F68,
pH 5.5); formulation F (-.DELTA.--) contains formulation E plus
0.9% BA; and formulation G (--.box-solid.--) contains formulation E
plus 0.25% phenol.
[0024] FIG. 12 depicts effect of preservative on Iso-aspartate
formation of rhNGF in liquid multi-dose formulations stored at 25
degrees C. for 9 months as determined by RP-HPLC. Formulation A
(-.crclbar.-) contains 2 mg/mL (10 mM acetate, pH 5.5); formulation
B (-.quadrature.-) contains 0.1 mg/mL (20 mM acetate, pH 5.5);
formulation C (--.diamond.--) contains formulation B plus 0.9% BA;
formulation D (--x--) contains formulation B plus 0.25% phenol;
formulation E (--+--) contains 0.1 mg/mL (20 mM acetate, 0.01% F68,
pH 5.5); formulation F (-.DELTA.--) contains formulation E plus
0.9% BA; and formulation G (--.box-solid.--) contains formulation E
plus 0.25% phenol.
[0025] FIG. 13 depicts cation exchange HPLC chromatograms of 0.1
mg/ml rhNGF in 10 mM acetate at pH 5.5 and 142 mM NaCl stored at 5
degrees C. (solid line), 25 degrees C. (dashed line), and 40
degrees C. (dotted line) for 3 months. Peak (a) contains mono and
di-oxidized 118/118 and oxidized N-terminally clipped rhNGF; peak
(b) contains 118/118 rhNGF homodimer; and peak (c) contains Ser-Gly
118/118 rhNGF (1-chain).
[0026] FIG. 14 depicts determination of rhNGF dimer (118/118)
remaining in rhNGF formulations after 12 months at 5 degrees C. by
cation exchange HPLC. Formulation A (-.crclbar.-) contains 2 mg/mL
(10 mM acetate, pH 5.5); formulation B (-.quadrature.-) contains
0.1 mg/mL (20 mM acetate, pH 5.5); formulation C (--.diamond.--)
contains formulation B plus 0.9% BA; formulation D (--x--) contains
formulation B plus 0.25% phenol; formulation E (--+--) contains 0.1
mg/mL (20 mM acetate, 0.01% F68, pH 5.5); formulation F
(-.DELTA.--) contains formulation E plus 0;9% BA; and formulation G
(--.circle-solid.--) contains formulation E plus 0.25% phenol.
[0027] FIG. 15 depicts determination of rhNGF dimer (118/118)
remaining in rhNGF formulations after 9 months at 25 degrees C. by
cation exchange HPLC. Formulation A (-.crclbar.-) contains 2 mg/mL
(10 mM acetate, pH 5.5); formulation B (-.quadrature.-) contains
0.1 mg/mL (20 mM acetate, pH 5.5); formulation C (--.diamond.--)
contains formulation B plus 0.9% BA; formulation D (--x--) contains
formulation B plus 0.25% phenol; formulation E (--+--) contains 0.1
mg/mL (20 mM acetate, 0.01% F68, pH 5.5); formulation F
(-.DELTA.--) contains formulation E plus 0.9% BA; and formulation G
(--.circle-solid.--) contains formulation E plus 0.25% phenol.
[0028] FIG. 16 depicts near UV CD spectrum of rhNGF in 10 mM
acetate, 136 mM NaCl, pH 5.5.
[0029] FIG. 17 depicts a comparison of near-UV CD spectra of rhNGF
in the presence (solid line) and absence (dotted line) of 0.9%
benzyl alcohol in 20 mM acetate at pH 5.5 and 136 mM NaCl after 24
hours at 25 degrees C.
DETAILED DESCRIPTION
[0030] The present invention is based on the discovery that NGF
formulated in pharmaceutically acceptable acetate buffer from pH 5
to pH 6 as a pharmaceutical composition has markedly increased
stability in these compositions. Acetate concentrations can range
from 0.1 to 200 mM, more preferably from 1 to 50 mM, and even more
5 to 30 mM, and most preferably from 10 to 20 mM. One preferred
embodiment has 20 mM acetate and another has 10 mM acetate in the
administered solution. A preferred acetate salt for enhancing
stability and buffering capacity is sodium acetate. However other
physiologically acceptable acetate salts can be used, for example
potassium acetate. Suitable pH ranges for the preparation of the
compositions herein are from 5 to 6, preferably 5.4 to 5.9, more
preferably 5.5 to 5.8. A preferred pH is 5.5 which enhances
stability and buffering capacity. Another preferred embodiment is
pH 5.8, A "pharmaceutically effective amount" of NGF refers to that
amount which provides therapeutic effect in various administration
regimens. The compositions herein are prepared containing amounts
of NGF from 0.07 to 20 mg/ml, preferably 0.08 to 15 mg/ml, more
preferably 0.09 to 10 mg/ml, and most preferably 0.1 to 2 mg/ml. In
a preferred embodiment the NGF concentration is 0.1 mg/ml. In
another preferred embodiment the NGF concentration is 2.0 mg/ml.
For use of these compositions in administration to human patients
suffering from peripheral neuropathies, for example, these
compositions may contain from about 0.1 mg/ml to about 2 mg/ml NGF,
corresponding to the currently contemplated dosage rate for such
treatment. NGF is well-tolerated and higher doses can be
administered if necessary as determined by the physician.
[0031] Optionally, but preferably, the formulation contains a
pharmaceutically acceptable salt, preferably sodium chloride, and
preferably at about physiological concentrations. Low
concentrations are preferred, e.g., less than about 0.3 M to about
0.05 M, preferably from 0.16 to 0.20 M NaCl, more preferably 0.13
to 0.15 M. In a preferred embodiment the sodium chloride
concentration is 136 mM. In another preferred embodiment the
concentration is. 142 mM.
[0032] Optionally, the formulations of the invention can contain a
pharmaceutically acceptable preservative. In some embodiments the
preservative concentration ranges from 0.1 to 2.0%, typically v/v.
Suitable preservatives include those known in the pharmaceutical
arts. Benzyl alcohol, phenol, m-cresol, methylparaben, and
propylparaben are preferred preservatives. Benzyl alcohol is a
particularly preferred preservative that results in enhanced NGF
stability. A particularly preferred benzyl alcohol concentration is
0.7 to 1.2%, more preferably 0.8 to 1.0%, with a particularly
preferred concentration of 0.9%.
[0033] Optionally, the formulations of the invention can include a
pharmaceutically acceptable surfactant. Preferred surfactants are
non-ionic detergents. Preferred surfactants include Tween 20 and
pluronic acid (F68). F68 is particularly preferred for enhancing
NGF stability. Suitable surfactant concentrations are 0.005 to
0.02%. A preferred concentration for surfactant is 0.01%.
Surfactants are used to minimize particulate formation.
[0034] In a particularly preferred embodiment the composition
contains an NGF concentration of 0.1 mg/ml, a sodium acetate
concentration of 20 mM, pH 5.5, a sodium chloride concentration of
136 mM, and benzyl alcohol concentration at 0.9% (v/v). In another
embodiment the NGF concentration is 2.0 mg/ml, the sodium acetate
concentration is 10 mM, pH 5.5, and the sodium chloride
concentration is 142 mM.
[0035] In another embodiment of the invention is provided a kit for
NGF administration, which includes a vial or receptacle containing
a pharmaceutical composition of the invention comprising a
pharmaceutically effective amount of nerve growth factor and a
pharmaceutically acceptable acetate-containing buffer. A preferred
vial volume is one suitable for multi-dose use--allowing repeated
withdrawal of sample. The increased stability attained with the
formulations of the invention allow multi-dose liquid formulation.
Typically a multi-dose vial will provide sufficient formulation to
supply sufficient dosage for one patient for one month, preferably
one week. For example, the composition volume generally ranges from
0.3 to 10.0 ml and more preferably from 1.6 to 2.0 ml, depending on
dose concentration, frequency and ease of use. For example, a
volume of 1.8 ml is convenient when either 0.3 ug/kg or 0.1 ug/kg
are used, allowing 7 or 24 doses, respectively. When a light
sensitive component, such as benzyl alcohol is present, the vial is
protected from intense light. Generally it is sufficient to store
the vial in a darkened refrigerator or within an opaque box.
However, the vial walls can comprise light transmission reducing
materials. For example, translucent amber or brown vials or an
opaque vail can be used. In preferred embodiments the vial contains
multi-dose formulation. For a vial configuration, a selected
multi-dose liquid formulation can be filled in 3 cc Type I glass
vial with 1.8 mL fill volume. Selection of stopper will be based on
compatibility of different types of stopper with the selected
formulation.
[0036] Compositions of the invention are typically stored at 2 to 8
degrees C. The formulations are stable to numerous freeze thaw
cycles as shown herein.
[0037] In another embodiment the formulation is prepared with the
above acetate concentrations.
[0038] A preferred means of preparing a formulation is to dialyze a
bulk NGF solution into the final formulation buffer. Final NGF
concentrations are achieved by appropriate adjustment of the
formulation with formulation buffer absent NGF. Also provided are
methods for the preparation of the composition of claim 1
comprising the steps of compounding said NGF and acetate-containing
buffer. Also provided are methods of increasing the stability of
NGF in a pharmaceutical composition containing NGF as active
principle, comprising incorporating acetate in said composition,
wherein said acetate is present in an amount and pH effective to
increase the stability of the NGF.
[0039] The compositions hereof including lyophilized forms, are
prepared in general by compounding the components using generally
available pharmaceutical compounding techniques, known per se.
Likewise, standard lyophilization procedures and equipment
well-known in the art are employed. A particular method for
preparing a pharmaceutical composition of NGF hereof comprises
employing purified (according to any standard protein purification
scheme) NGF, preferably rhNGF, in any one of several known buffer
exchange methods, such as gel filtration or dialysis.
[0040] Nerve growth factor ("NGF") is a 120 amino acid polypeptide
homodimeric protein that has prominent effects on developing
sensory and sympathetic neurons of the peripheral nervous system.
NGF acts via specific cell surface receptors on responsive neurons
to support neuronal survival, promote neurite outgrowth, and
enhance neurochemical differentiation. NGF actions are accompanied
by alterations in neuronal membranes, in the state of
phosphorylation of neuronal proteins, and in the abundance of
certain mRNAs and proteins likely to play a role in neuronal
differentiation and function. (Connolly et al., J. Cell. Biol.
90:176-180 [1981]; Skaper and Varon, Brain Res. 197:379-389 [1980];
Yu, et al., J. Biol. Chem. 255:10481-10492 [1980]; Haleqoua and
Patrick, Cell 22:571-581 [1980]; Tiercy and Shooter, J. Cell. Biol.
103:2367-2378 [1986]).
[0041] Forebrain cholinergic neurons also respond to NGF and may
require NGF for trophic support. (Hefti, J. Neurosci., 6: 2155
[1986]). Indeed, the distribution and ontogenesis of NGF and its
receptor in the central nervous system (CNS) suggest that NGF acts
as a target-derived neurotrophic factor for basal forebrain
cholinergic neurons (Korsching, TINS, pp 570-573 [November/December
1986]).
[0042] Little is known about the NGF amino acid residues necessary
for the interaction with the trkA-tyrosine kinase receptor.
Significant losses of biological activity and receptor binding were
observed with purified homodimers of human and mouse NGF,
representing homogenous truncated forms modified at the amino and
carboxy termini. The 109 amino acid species (10-118)hNGF, resulting
from the loss of the first 9 residues of the N-terminus and the
last two residues from the C-terminus of purified recombinant human
NGF, is 300-fold less efficient in displacing mouse [.sup.125I]NGF
from the human trkA receptor compared to (1-118)hNGF. It is 50- to
100-fold less active in dorsal root ganglion and sympathetic
ganglion survival compared to (1-118)hNGF. The (1-118)hNGF has
considerably lower trkA tyrosine kinase autophosphorylation
activity. A preferred form is the 118 amino acid human NGF, which
is more preferable as a homodimer.
[0043] The formulations of the invention include the pantropic
neurotrophin pantropic NGF. Pantropic NGF is a pantropic
neurotrophin which has an amino acid sequence homologous to the
amino acid sequence of NGF, with domains which confer other
neurotrophin specificities. In the preferred embodiment, the
domains are substituted for NGF residues; that is, some number of
amino acids are deleted from the NGF sequence, and an identical or
similar number of amino acids are substituted, conferring an
additional specificity. For example, a pantropic NGF is made with a
D16A substitution, which confers BDNF specificity. Optionally,
substitutions in the pre-variable region 1 (V18E+V20L+G23T) and in
variable region 4 (Y79Q+T81K.sup.+ H84Q+F86Y+K88R) are included.
Alternatively, the substitutions in the pre-variable region 1 can
be made with only single amino acid substitutions in variable
region 4; for example, V18E+V20L+G23T and one of Y79Q, T81K, H84Q,
F86Y, or K88R may be made.
[0044] The chemical and physical stability of recombinant human
nerve growth factor (NGF) in aqueous solution was investigated
between 5 and 37.degree. C., in the pH range 4.2 to 5.8. NGF
chemical stability increased with increasing pH. In succinate
buffer at pH 5.8, NGF physical stability decreased due to protein
aggregation. Based on both the 5.degree. C. stability data and
accelerated degradation studies at 37.degree. C., the optimal
formulation was found to be acetate buffer at pH 5.8.
Reversed-phase HPLC was the primary stability indicating method,
showing conversion of Asn-93 to iso-Asp to be the primary
degradation pathway at 5.degree. C. Quantitation of NGF degradation
by cation exchange chromatography was complicated by the
rearrangement of the NGF monomer variants into various mixed dimers
over time (dimer exchange). Treatment of samples and controls with
dilute acid rapidly equilibrated the monomer distribution in the
dimers, allowing NGF degradation to be quantitated in the absence
of dimer exchange.
[0045] Benzyl alcohol and phenol were evaluated for their
compatibility and stability with rhNGF in two liquid formulations
for multi-use purposes. These two formulations consist of 0.1 mg/mL
protein in 20 mM sodium acetate at pH 5.5 and 136 mM sodium
chloride with and without 0.01% pluronic acid (F68) as surfactant.
The final concentrations of benzyl alcohol and phenol in each of
these two formulations were 0.9 and 0.25%, respectively. Based on
the 12 month stability data, rhNGF is more stable with benzyl
alcohol than phenol in these formulations. Benzyl alcohol preserved
rhNGF formulation with the presence of surfactant is as stable as
the formulation with no surfactant added, indicating that the
addition of F68 to rhNGF multi-dose formulation is not required for
stability purpose. Therefore, a formulation consisting of 0.1 mg/mL
protein in 20 mM acetate, 136 mM NaCl, 0.9% benzyl alcohol*, pH 5.5
is recommended for rhNGF used for multiple dosing in Phase III
clinical trails. This rhNGF multi-dose formulation passed the USP
and EP preservative efficacy test after 6 months at 5 degrees C.,
and is as stable as the current liquid formulation at 2 mg/mL.
However, the formulation should avoid exposure to intensive light
due to the presence of benzyl alcohol as preservative which is
light sensitive.
[0046] In general, the compositions may contain other components in
amounts preferably not detracting from the preparation of stable,
liquid or lyophilizable forms and in amounts suitable for
effective, safe pharmaceutical administration.
[0047] In order that materials like NGF be provided to health care
personnel and patients, these materials must be prepared as
pharmaceutical compositions. Such compositions must be stable for
appropriate periods of time, must be acceptable in their own right
for administration to humans, and must be readily manufacturable.
An example of such a composition would be a solution designed for
parenteral administration. Although in many cases pharmaceutical
solution formulations are provided in liquid form, appropriate for
immediate use, such parenteral formulations may also be provided in
frozen or in lyophilized form. In the former case, the composition
must be thawed prior to use. The latter form is often used to
enhance the stability of the medicinal agent contained in the
composition under a wider variety of storage conditions, as it is
recognized by those skilled in the art that lyophilized
preparations are generally more stable than their liquid
counterparts. Such lyophilized preparations are reconstituted prior
to use by the addition of suitable pharmaceutically acceptable
diluent(s), such as sterile water for injection or sterile
physiological saline solution, and the like.
[0048] NGF formulations of the invention are believed to be useful
in promoting the development, maintenance, or regeneration of
neurons in vivo, including central (brain and spinal chord),
peripheral (sympathetic, parasympathetic, sensory, and enteric
neurons), and motorneurons. Accordingly, NGF formulations of the
invention are utilized in methods for the treatment of a variety of
neurologic diseases and disorders. In a preferred embodiment, the
formulations of the present invention are administered to a patient
to treat neural disorders. By "neural disorders" herein is meant
disorders of the central and/or peripheral nervous system that are
associated with neuron degeneration or damage. Specific examples of
neural disorders include, but are not limited to, Alzheimer's
disease, Parkinson's disease, Huntington's chorea, stroke, ALS,
peripheral neuropathies, and other conditions characterized by
necrosis or loss of neurons, whether central, peripheral, or
motorneurons, in addition to treating damaged nerves due to trauma,
burns, kidney disfunction, injury, and the toxic effects of
chemotherapeutics used to treat cancer and AIDS. For example,
peripheral neuropathies associated with certain conditions, such as
neuropathies associated with diabetes, AIDS, or chemotherapy may be
treated using the formulations of the present invention. It also is
useful as a component of culture media for use in culturing nerve
cells in vitro or ex vivo.
[0049] In various embodiments of the invention, NGF formulations
are administered to patients in whom the nervous system has been
damaged by trauma, surgery, stroke, ischemia, infection, metabolic
disease, nutritional deficiency, malignancy, or toxic agents, to
promote the survival or growth of neurons, or in whatever
conditions have been found treatable with NGF. For example, NGF
formulation of the invention can be used to promote the survival or
growth of motorneurons that are damaged by trauma or surgery. Also,
NGF formulations of the invention can be used to treat motoneuron
disorders, such as amyotrophic lateral sclerosis (Lou Gehrig's
disease), Bell's palsy, and various conditions involving spinal
muscular atrophy, or paralysis. NGF formulations of the invention
can be used to treat human neurodegenerative disorders, such as
Alzheimer's disease, Parkinson's disease, epilepsy, multiple
sclerosis, Huntington's chorea, Down's Syndrome, nerve deafness,
and Meniere's disease. NGF formulations of the invention can be
used as cognitive enhancer, to enhance learning particularly in
dementias or trauma. Alzheimer's disease, which has been identified
by the National Institutes of Aging as accounting for more than 50%
of dementia in the elderly, is also the fourth or fifth leading
cause of death in Americans over 65 years of age. Four million
Americans, 40% of Americans over age 85 (the fastest growing
segment of the U.S. population), have Alzheimer's disease.
Twenty-five percent of all patients with Parkinson's disease also
suffer from Alzheimer's disease-like dementia. And in about 15% of
patients with dementia, Alzheimer's disease and multi-infarct
dementia coexist. The third most common cause of dementia, after
Alzheimer's disease and vascular dementia, is cognitive impairment
due to organic brain disease related directly to alcoholism, which
occurs in about 10% of alcoholics. However, the most consistent
abnormality for Alzheimer's disease, as well as for vascular
dementia and cognitive impairment due to organic brain disease
related to alcoholism, is the degeneration of the cholinergic
system arising from the basal forebrain (BF) to both the codex and
hippocampus (Bigl et al. in Brain Cholinergic Systems, M. Steriade
and D. Biesold, eds., Oxford University Press, Oxford, pp.364-386
(1990)). And there are a number of other neurotransmitter systems
affected by Alzheimer's disease (Davies Med Res. Rev.3:221 (1983)).
However, cognitive impairment, related for example to degeneration
of the cholinergic neurotransmitter system, is not limited to
individuals suffering from dementia. It has also been seen in
otherwise healthy aged adults and rats. Studies that compare the
degree of learning impairment with the degree of reduced cortical
cerebral blood flow in aged rats show a good correlation (Berman et
al. Neurobiol. Aging 9:691 (1988)). In chronic alcoholism the
resultant organic brain disease, like Alzheimer's disease and
normal aging, is also characterized by diffuse reductions in
cortical cerebral blood flow in those brain regions where
cholinergic neurons arise (basal forebrain) and to which they
project (cerebral cortex) (Lofti et al., Cerebrovasc. and Brain
Metab. Rev 1:2 (1989)). Such dementias can be treated by
administration of NGF formulations of the invention.
[0050] Further, NGF formulations of the invention are preferably
used to treat neuropathy, and especially peripheral neuropathy.
"Peripheral neuropathy" refers to a disorder affecting the
peripheral nervous system, most often manifested as one or a
combination of motor, sensory, sensorimotor, or autonomic neural
dysfunction. The wide variety of morphologies exhibited by
peripheral neuropathies can each be attributed uniquely to an
equally wide number of causes. For example, peripheral neuropathies
can be genetically acquired, can result from a systemic disease, or
can be induced by a toxic agent. Examples include but are not
limited to diabetic peripheral neuropathy, distal sensorimotor
neuropathy, or autonomic neuropathies such as reduced motility of
the gastrointestinal tract or atony of the urinary bladder.
Examples of neuropathies associated with systemic disease include
post-polio syndrome; examples of hereditary neuropathies include
Charcot-Marie-Tooth disease, Refsum's disease,
Abetalipoproteinemia, Tangier disease, Krabbe's disease,
Metachromatic leukodystrophy, Fabry's disease, and Dejerine-Sottas
syndrome; and examples of neuropathies caused by a toxic agent
include those caused by treatment with a chemotherapeutic agent
such as vincristine, cisplatin, methotrexate, or
3'-azido-3'-deoxythymidi- ne.
[0051] A therapeutically effective dose of an NGF formulation is
administered to a patient. By "therapeutically effective dose"
herein is meant a dose that produces the effects for which it is
administered. The exact dose will depend on the disorder to be
treated, and will be ascertainable by one skilled in the art using
known techniques. In general, the NGF formulations of the present
invention are administered at about 0.01 .mu.g/kg to about 100
mg/kg per day. Preferably, from 0.1 to 0.3 ug/kg. In addition, as
is known in the art, adjustments for age as well as the body
weight, general health, sex, diet, time of administration, drug
interaction and the severity of the disease may be necessary, and
will be ascertainable with routine experimentation by those skilled
in the art. Typically, the clinician will administer NGF
formulations of the invention until a dosage is reached that
repairs, maintains, and, optimally, reestablishes neuron function.
The progress of this therapy is easily monitored by conventional
assays.
[0052] A "patient" for the purposes of the present invention
includes both humans and other mammals. Thus the methods are
applicable to both human therapy and veterinary applications.
[0053] Therapeutic formulations of NGF are prepared by mixing NGF
having the desired degree of purity with optional physiologically
acceptable carriers, excipients or stabilizers (Remington's
Pharmaceutical Sciences). Acceptable carriers, excipients or
stabilizers are nontoxic to recipients at the dosages and
concentrations employed and will not significantly decrease NGF
stability in the formulations as taught herein. Such compounds
include antioxidants including ascorbic acid; low molecular weight
(less than about 10 residues) polypeptides; proteins, such as serum
albumin, gelatin or immunoglobulins; hydrophilic polymers such as
polyvinylpyrrolidone, amino acids such as histidine, methionine,
glycine, glutamine, asparagine, arginine or lysine;
monosaccharides, disaccharides and other carbohydrates including
glucose, mannose, or dextrins; chelating agents such as EDTA; sugar
alcohols such as mannitol or sorbitol; salt-forming counterions
such as sodium; and/or non-ionic surfactants such as Tween,
Pluronics or PEG.
[0054] NGF formulations to be used for in vivo administration must
be sterile. This is readily accomplished by filtration through
sterile filtration membranes. Ordinarily NGF formulations of the
present invention will be stored in liquid form at 2 to 8 degrees
C. The formulations are suitable for frozen storage with repeated
cycles of thawing and freezing.
[0055] Therapeutic NGF compositions generally are placed into a
container having a sterile access port, for example, an intravenous
solution bag or vial having a stopper pierceable by a hypodermic
injection needle.
[0056] NGF optionally is combined with or administered in concert
with other neurotrophic factors including NT-4/5, NT-3, and/or BDNF
and is used with other conventional therapies for nerve
disorders.
[0057] The administration of the formulations of the present
invention can be done in a variety of ways, including, but not
limited to, orally, subcutaneously, intravenously, intracerebrally,
intranasally, transdermally, intraperitoneally, intramuscularly,
intrapulmonary, vaginally, rectally, or intraocularly. The
formulations can be administered continuously by infusion into the
fluid reservoirs of the CNS, although bolus injection is
acceptable, using techniques well known in the art, such as pumps
or implantation. In some instances, for example, in the treatment
of wounds, the formulations may be directly applied as a solution
or spray.
[0058] The following examples are offered by way of illustration
and not by way of limitation. The disclosures of all citations in
the specification are expressly incorporated herein by
reference.
EXAMPLES
Example I
[0059] Materials
[0060] Recombinant human nerve growth factor (NGF) was produced in
Chinese hamster ovary cells and purified by reversed-phase
(RP-HPLC) and ion-exchange chromatography (IEC) as described
previously (8). HPLC grade acetonitrile, and TFA were used for
RP-HPLC. All other chemicals were USP grade. Sterile type I, clear
glass, 2 cc vials were purchased from Wheaton and used with
siliconized, Teflon-coated, butyl rubber stoppers.
[0061] Methods
[0062] NGF was dialyzed into 10 mM sodium acetate, 142 mM sodium
chloride, at pH 5.0 and 5.8, and into 10 mM sodium succinate, 142
mM NaCl, at pH 4.2, 5.0, and 5.8, and adjusted to 10 mg/ml. Tween
20 was also added to a succinate pH 5.0 formulation to determine if
surfactant would reduce NGF aggregation (10 mM sodium succinate,
142 mM NaCl, 0.05% Tween 20).
[0063] Vials were aseptically filled with 0.3 ml of NGF formulation
and stored at 5, 25, and 37.degree. C. (25.degree. C. data not
reported here). Controls were stored at -70.degree. C. where no
significant degradation has been observed. At each time point, 50
.mu.l aliquots were removed from individual vials and stored at
-70.degree. C. until analysis.
[0064] HPLC Analysis. Cation exchange HPLC (IEC) was carried out on
a HP 1090 system using a Tosohas sulpho-propyl TSK-SP-5PW
(7.5.times.75 mm) column with 10 m particles. Mobile phases were
(A) 10 mM sodium phosphate, 5% (v/v) acetonitrile, pH 7.0 and (B)
A+1.0 M ammonium chloride. NGF was eluted at 35.degree. C. (0.5
ml/min) with a linear gradient of 20-40% B from 5 to 60 minutes.
The control and 1.6 year samples at 5.degree. C. were also assayed
after "acid-treatment" to bring the distribution of monomer
variants in the dimers to equilibrium (8, 9). These samples were
adjusted to pH 3.5 with HCl and incubated at 37.degree. C. for 2
hours (results at 2 and 4 hours were equivalent). A YMC C4, 5 .mu.m
(4.6.times.250 mm) column was used for reversed-phase HPLC
(RP-HPLC) on a HP 1090 system at 25.degree. C. NGF was eluted (0.5
m/min) using a linear gradient of 26-30% B in A (B=0.05% TFA in
acetonitrile and A=0.05% TFA in water) run between 5 and 40
minutes. Size exclusion HPLC ("SEC-HPLC") was carried out using a
Perkin Elmer Series 410 Bio LC Pump with a Perkin Elmer LC 90
Spectrophotometric UV Detector and a Tosohas TSK 2000 SWXL, 5 .mu.m
(7.8.times.300 mm) column. This SEC column was run at 0.5 ml/min
using a 0.2 M potassium phosphate, 0.45 M potassium chloride mobile
phase, at pH 7.0. For SEC UV detection was at 280 nm; for RP-HPLC
and IEC, at 214 nm. For all assays 50 mg of NGF were injected.
[0065] SDS-PAGE. Samples were diluted into Novex tricine SDS sample
buffer and incubated for 1 hour at 50.degree. C. Non-reduced
SDS-PAGE was run on Novex tricine gels containing 10% acrylamide
followed by Coomassie Blue staining. Molecular weights were
estimated using Bio-Rad low molecular weight markers. Neurite
Outgrowth Assay. The biological activity of NGF was determined
using the PC 12 assay developed by Greene (10) and modified as
described by Schmelzer et al (8).
[0066] Hemolysis. All formulations were tested for hemolytic
activity. The hemolysis procedure was that of Reed and Yalkowsky
(11) except that equal volumes of washed human red blood cells and
formulation were incubated at 37.degree. C. for 30 minutes before
analysis.
[0067] Results
[0068] Formulation development of NGF requires condition be found
for which the protein shows .gtoreq.1.5 years of chemical and
physical stability at 2-8.degree. C. We determined the approximate
pH of maximal NGF stability by ascertaining NGF stability in
succinate buffer at pH 4.2, 5.0, and 5.8, and acetate buffer at pH
5.0 and 5.8. NGF stability decreases above pH 6.0. The assays used
to measure protein stability were IEC, SEC, RP-HPLC, SDS-PAGE, and
the PC12 bioactivity assay. Formulation biocompatibility was
determined by hemolysis testing.
[0069] Stability of NGF at 37.degree. C.
[0070] Aggregation of NGF. The dimer/monomer equilibrium constant
for murine NGF is smaller than 10-13 M at pH 4-7 (6, 7, 9, 12).
NGF, therefore, assayed primarily as a dimer in the neutral pH SEC
assay. A small amount of aggregated NGF (tetramer based on
molecular weight standards) was observed in the control sample.
This tetramer peak area increased with time at 37.degree. C. A
leading shoulder on this peak, indicating larger aggregates, was
observed for all formulations after 38 days at 37.degree. C. The
time dependencies of aggregate formation for the various
formulations are shown in FIG. 1. The succinate pH 5.8 formulation
had the greatest aggregation rate. All other formulations had
similar rates of aggregate formation. The addition of the
surfactant Tween 20 offered no protection against aggregation in
the pH 5.0 succinate formulation. During preparation, the NGF pH
5.8 succinate formulation had to be filtered through a 47 mm
diameter 0.22 mm filter, whereas all other formulation were
filterable through a 25 mm diameter filter. This is consistent with
the high rate of aggregation observed at 37.degree. C. in succinate
buffer at pH 5.8.
[0071] Aggregation was also monitored using non-reduced SDS-PAGE
(gels not shown). In the -70.degree. C. control samples 3 bands
were observed: monomer at 13.5 kDa, a very faint dimer band at
approximately 26 kDa, and a slightly more intense band at 31 kDa.
The 26 and 31 kDa bands became more intense on incubation at
elevated temperatures. A small amount of large molecular weight
aggregate (>97 kDa) was observed in all formulations after 38
days at 37.degree. C. The intensity of this band was greatest in
the pH 5.8 succinate formulation, consistent with the poor
filterability and high aggregation rate observed by SEC for this
formulation. Tween 20 prevented the formation of this high
molecular weight aggregate at pH. 5.0. With the exception of
succinate at pH 5.8, these sizing methods do not differentiate
between the quality of the NGF formulations.
[0072] NGF Monomer and Degradation Product Quantitation. The NGF
used in these studies consisted of a 1:9:1 ratio of the three
monomeric polypeptides containing 120, 118, and 117 amino acids.
The 118 amino acid variant was produced by clipping of Ala120 and
Arg119 from the C-terminus of the 120 parent; the 117 variant had
an additional clip, Arg118 (8). At pH 5.0, the 117 variant has two
fewer positive charges, and the 118 variant one fewer positive
charge than the 120 parent. There is no significant difference in
the bioactivity of the homodimers and heterodimers formed by the
117, 118, and 120 variants as measured by the PC12 and chick dorsal
root ganglion assays (8). In the acidic, organic, RP mobile phase
where NGF dissociates to monomer (8), the elution order is 120
before 118, then 117. Typical RP-HPLC chromatograms for NGF stored
in pH 5.0 succinate buffer, for 38 days, at -70.degree. C. and
37.degree. C. are shown in FIG. 2. At elevated temperature, peak
area is lost from the peaks defined as NGF (the sum of the 118 and
120 monomer peaks) with the iso-Asp, oxidized, and other NGF
degradation peaks increasing in area. The 117 peak area was not
included in the definition of NGF due to coelution of degradation
products with this peak at elevated temperatures. The time
dependence of NGF degradation at 37.degree. C., and the apparent
first order rate constants for this degradation, are shown in FIG.
3 and Table 1, respectively.
1TABLE 1 Apparent First-Order Rate Constants for NGF Degradation at
37.degree. C. as Determined by RP-HPLC. Buffer pH k (day-1)
Succinate 4.2 2.2 .times. 10.sup.-2 .+-. 1.0 .times. 10.sup.-3 5.0
1.1 .times. 10.sup.-2 .+-. 6.3 .times. 10.sup.-4 (+Tween 20) 5.0
1.1 .times. 10.sup.-2 .+-. 7.1 .times. 10.sup.-4 5.8 5.7 .times.
10.sup.-3 .+-. 9.7 .times. 10.sup.-4 Acetate 5.0 7.9 .times.
10.sup.-3 .+-. 8.0 .times. 10.sup.-4 5.8 4.0 .times. 10.sup.-3 .+-.
2.9 .times. 10.sup.-4
[0073] NGF stability decreased as the pH was lowered. In both the
acetate and succinate pH 5.8 buffers NGF stability was greater than
at pH 5.0. In succinate buffer at pH 4.2, the NGF degradation rate
is further increased, with several hydrophobic degradation products
being observed, possibly due to acid-induced cleavage at the
Asp60-Pro61 linkage. Tween 20 had no affect on NGF stability in
succinate buffer at pH 5.0 (FIG. 3). The acetate formulation
appears to be somewhat better in maintaining NGF stability.
[0074] NGF Dimer Distribution. The three NGF monomers containing
117, 118, and 120 amino acids may combine to form the 117/117,
118/118 and 120/120 homodimers and the 117/118, 118/120, and
117/120 heterodimers. Association of these NGF variants has been
shown to be random, with no monomer appearing to prefer any other
(8, 9). The dynamic dissociation and reassociation of monomers to
form various dimers (dimer exchange) is accelerated by low pH and
increased temperature (9). For a random association process at
equilibrium, and an initial 117/118/120 ratio of 1:9:1, the 118/118
homodimer will be the dominant dimer species with smaller amounts
of the 117/118 and 118/120 dimers being formed.
[0075] The 118/118 and 117/120 dimers have the same effective net
charge in the chosen IEC mobile phase and therefore coelute on IEC
during NGF purification. This results in an initial non-equilibrium
distribution of the monomer variants in NGF dimers in the NGF
product. The 117/120 and 118/118 dimers dissociate giving the 117
and 120 monomers which will reassociate most frequently with 118
monomer to form 117/118 and 118/120 dimers. Due to the different
charges on the monomers, the expected elution order of these dimers
on cation-exchange chromatography is:
[0076]
117/117<117/118<118/118=117/120<118/120<120/120.
[0077] The most populated dimers are distinguishable by IEC (8) as
shown in FIG. 4.
[0078] Representative IEC chromatograms for NGF at pH 5.0 in
succinate buffer after 38 days at -70.degree. C. and 37.degree. C.
are shown in FIG. 4. During NGF production, a fraction of the
N-terminal serine residues are converted to glycine with no affect
on NGF activity (13). NGF is quantitated here as the sum of the
118/118 homodimer and the 118/118 dimer with a Ser1 to Gly1
conversion in one of the two monomers (13) (and any coeluting
117/120 variants); the 117/118 and 118/120 peak areas are not
included due to degradation products coeluting with these peaks.
The rate of loss of NGF, as monitored by IEC at 37.degree. C., is
shown in FIG. 5. The degradation kinetics for the 118 dimer are
multiphasic. The loss in main peak area before 13 days is largely
due to rearrangement of the monomer variants between the possible
dimer types. The data after 13 days more accurately describes NGF
chemical degradation. NGF is most stable in the acetate
formulations at pH 5.0 and 5.8, which have similar stability. NGF
in succinate buffer at pH 5.8, and pH 5.0, with and without 0.05%
Tween 20, all have similar stabilities. The hemolytic activity of
each of the NGF formulations was also tested. None of the
formulations showed significant red blood cell hemolysis
(<0.1%). The bioactivity of NGF in each of the formulations was
also determined, using the neurite extension PC12 assay. NGF was
bioactive in all of the formulations after 38 days at 37.degree. C.
The large assay variability (approximately 50% error) did not allow
quantitative bioactivity differences between these formulations to
be determined.
[0079] A liquid formulation for NGF preferably has an adequate
shelf-life at 5.degree. C. The accelerated stability data at
37.degree. C. showed NGF to be most stable in acetate buffer. Based
on this data, NGF stability in the acetate pH 5.0 and 5.8
formulations was investigated for 1.6 years at 5.degree. C. RP-HPLC
chromatograms at pH 5.0 for the 1.6 year -70.degree. C. control and
5.degree. C. samples are shown in FIG. 6. The major degradation
product was Asp-93 conversion to iso-Asp; smaller amounts of Met-37
and Met-92 oxidation were observed. The apparent first order rate
constants for NGF degradation, quantitated by RP-HPLC, are
1.4.times.10-4.+-.1.7.times.10-5 d-1 and
6.8.times.10-5.+-.7.0.times.10-6 d-1 at pH 5.0 and 5.8,
respectively. At 5.degree. C., IEC shows that NGF stability is
approximately the same at pH 5.0 and 5.8, consistent with the
37.degree. C. IEC data. Aggregation of the NGF dimers was not a
significant degradation pathway at 5.degree. C., only a 1% increase
in aggregate was observed over 1.6 years of storage at 5.degree.
C.
[0080] The interpretation of the IEC data at both 5.degree. C. and
37.degree. C., is complicated by dimer exchange, the exchange rate
being slower at the lower temperature. To improve IEC quantitation,
the dimer distribution was brought to equilibrium by incubation at
pH 3.5 for 2 hours at 37.degree. C. prior to IEC analysis (8,9,14).
No new degradation products were observed after this treatment. The
acetate pH 5.8 samples after 1.6 years of incubation at 5.degree.
C. are compared with controls before and after "acid treatment" in
FIG. 7. The loss of main peak area to the peripheral peaks due to
dimer exchange was eliminated by acid treatment, revealing the true
degradation of NGF. Quantitation after acid treatment showed that
94 and 92% of the NGF main peaks remain after 1.6 years at
5.degree. C. at pH 5.0 and pH 5.8, respectively, compared to 84 and
87% without acid treatment. For comparison, RP-HPLC analysis showed
93 and 96% of the NGF 118 and 120 monomers remaining at pH 5.0 and
pH 5.8, respectively.
[0081] NGF chemical stability was shown to increase with pH, the pH
of maximal stability being near pH 5.8. At a fixed pH, the RP-HPLC
and IEC data at 5 and 37.degree. C. were consistent in showing NGF
chemical stability to be greater in acetate than succinate buffer.
In addition, NGF aggregation was not a significant degradation
pathway, except at pH 5.8 in succinate buffer. A complicating
factor in the determination of NGF stability is that dimer exchange
contributes to the apparent degradation of NGF dimers as determined
by IEC. A more accurate representation of NGF chemical degradation
can be obtained by pretreating the controls and samples with acid
at 37.degree. C. to bring the dimer distribution to equilibrium.
Taken together, these data show that the optimal formulation and
storage conditions for NGF stability are acetate butter at pH 5.8
with storage at 5.degree. C.
Example II
[0082] Results from Phase II clinical trials indicate that patients
with peripheral neuropathy disease require three dosings per week
of rhNGF at either 0.3 or 0.1 .mu.g/kg. This means that only 21 or
7 .mu.g per dosing of rhNGF is needed for an average patient of
body weight 70 kg. Using the current rhNGF liquid formulation (2
mg/mL in 10 mM sodium acetate, pH 5.5, 142 mM NaCl) and vial
configuration (0.7 mL per vial) would have wasted a lot of drug
product. Therefore, a new rhNGF formulation at low concentration,
preferably multi-dose configuration, is required to reduce the cost
and wastage of the product. The purpose of this study was to
develop a stable multi-dose liquid formulation for rhNGF at 0.1
mg/mL with 1.8 mL fill in 3 cc glass vial for use in Phase III
clinical trails. With this new configuration, each vial will give
180 .mu.g protein and will provide at least 7 doses at the high
dosing level (0.3 .mu.g/kg) and 24 doses at the low dosing level
(0.1 .mu.g/mL).
[0083] In this study, the results on compatibility and stability of
preservative containing 0.1 mg/ml rhNGF multi-dose liquid
formulations at pH 5.5 are presented. A comparison between the
stability of the new multi-dose liquid formulations at 0.1 mg/mL
rhNGF and the current 2 mg/mL rhNGF formulation was also studied.
Results on agitation, freezing and thawing, and light compatibility
studies of the lead multi-dose liquid formulations for 0.1 mg/mL
rhNGF were also reported.
[0084] In this study, rhNGF concentrated bulk formulated at 11.6
mg/mL in 10 mM sodium acetate, 142 mM sodium chloride at pH 5.5
with 20 mL filled in 100 cc glass vials was used. All chemical
reagents and materials used in this Example are listed in Table
2.
2TABLE 2 List of Materials rhNGF concentrated bulk, 11.6 mg/mL, in
10 mM sodium acetate, 142 mM sodium chloride, pH 5.5 Sodium acetate
trihydrate, Genentech Release Materials Code G20136, Lot #S0766
Glacial acetic acid, Release Materials Code G20027-01, Lot S0567
Sodium Chloride, Release Materials Code G20136, Lot S1152 Benzyl
alcohol, Release Materials Code G20226, Lot C0200 m-cresol, Sigma,
Lot 107F-3497 Methylparaben, Napp Chemical Inc., Lot LM 86-6285
Propylparaben, Napp Chemical Inc., LL86-6241 Phenol, Release
Materials Code G20136, Lot 620015, Lot B0901 Polysorbate 20,
Release Materials Code G20091, Lot A1408 Pluronic acid (F68),
Release Materials Code GXXXX, Lot XXXX Sterile, pryogen-free
non-siliconized Type I clear glass 3 cc vials (Wheaton Tubing
Products); prepared in Phase V per standard procedures Sterile 13
mm Purcoat rubber stoppers, Clinical manufacturing, Genentech, Inc.
13 mm aluminum flip-off cap, Clinical manufacturing, Genentech,
Inc.
[0085] Methods
[0086] rhNGF Multi-dose Liquid Formulations Preparation. rhNGF
concentrated bulk was dialyzed into a formulation buffer consisting
of 20 mM sodium acetate, 136 mM sodium chloride at pH 5.5 by
ultrafiltration using Amicon Centriprep.TM. concentrator with
molecular weight cutoff of 10,000 KD. This reformulated rhNGF bulk
was then diluted to 0.15 mg/mL using the same formulation buffer
for dialysis. Preservatives and surfactants used for compatibility
screening and formulation development studies were added to this
diluted rhNGF solution at their tested concentrations. Protein
concentration for each formulation was then adjusted to 0.1 mg/mL
by UV analysis using the appropriate formulation buffer. A list of
preservatives and their concentrations used for physical
compatibility with rhNGF in liquid formulations are given in table
3.
3TABLE 3 List of Preservative Screening Formulations for 0.1 mg/mL
rhNGF Formulation buffer Surfactant Preservative 20 mM acetate, pH
5.5 none 0.9% benzyl alcohol 136 mM NaCl 0.25% phenol 0.45% phenol
0.25% m-cresol 0.18% methylparaben 0.02% propylparaben 20 mM
acetate, pH 5.5 0.01% Tween 20 0.9% benzyl alcohol 136 mM NaCl
0.25% phenol 0.45% phenol 0.25% m-cresol 0.18% methylparaben 0.02%
propylparaben 20 mM acetate, pH 5.5 0.01% F68 0.9% benzyl alcohol
136 mM NaCl 0.25% phenol 0.45% phenol 0.25% m-cresol 0.18%
methylparaben 0.02% propylparaben
[0087] Experimental Design
[0088] All rhNGF multi-dose liquid formulations prepared were
sterile filtered through 0.22 .mu.m filter prior to filling. Each
formulations were aseptically filled into Type 1, clear glass, 3 cc
Wheaton vials with a fill volume of 1.8 mL. Vials were stoppered
with 13 mm Purcoat stoppers and hand crimped with 13 mm aluminum
flip-off caps.
[0089] For the preservative screening study, samples were stored at
room temperature for 24 hours to determine physical compatibility.
For the formulation development study, samples were stored at -70,
5, 25 and 40.degree. C. At each time point, one
sample/formulation/temperature was assayed.
[0090] Agitation studies were carried out at room temperature on
the current 2 mg/mL rhNGF formulation, the multi-dose formulations
that contain either 0.9% benzyl alcohol or 0.25% phenol in the
absence of surfactant, and the 0.1 mg/mL rhNGF control that
contains no 24. surfactant and preservative. A 3 cc vial of each
formulation tested was secured to a laboratory bench top shaker
(Glas-Col) and agitated at 80 rpm for 6 and 24 hours. Samples
collected after 6 and 24 hours of shaking were assayed by SE-HPLC,
RP-HPLC, ELISA and RRA.
[0091] Freezing and thawing cycling was performed on the same
formulations that used for agitation studies. One vial from each
formulation tested was placed in -70.degree. C. freezer and allowed
to freeze for 24 hours. After 24 hours of freezing, samples were
thawed at 5.degree. C. for 24 hours. This freezing and thawing
procedure was repeated up to 3 times. Samples collected at the end
of the third cycle were assayed by SE-HPLC, RP-HPLC, ELISA and
RRA.
[0092] The effect of light on stability of rhNGF was studied on the
same formulations that used for agitation studies. One vial from
each formulation was placed in a light box (Form a Scientific,
Model 3890) under high intensity fluorescent light for 5 weeks.
Control vials wrapped with aluminum foil were also placed in the
light box. Light intensity was 20,000 lux which was about 15-20
times that of indoor fluorescent light, and the temperature of the
light box was maintained at 28.degree. C. Samples were assayed at 2
and 5 weeks by SEC-HPLC, ELISA and RRA.
[0093] Analytical Methodology
[0094] A. UV Analysis. rhNGF concentration was determined by
scanning from 240 to 360 nm using an HP 8452A UV-Vis
spectrophotometer. Formulation buffer was used as a reference to
blank the instrument, and the protein concentration in mg/mL was
calculated from (A280-320)/1.5, where 1.5 is the extinction
coefficient of rhNGF in mL/(mg.multidot.cm).
[0095] B. HPLC Analysis. The following HPLC methods were used.
Reversed-Phase HPLC
4 column: YMC C4, 5 .mu.m, 4.6 .times. 250 mm mobile phase: A:
0.05% (v/v) TFA, water B: 0.05% (v/v) TFA, 100% AcCN gradient:
25-27% B (26'), 27-50% B (4'), 50-80% B (1'), 80-25% B (4'), 25% B
(20') flow rate: 1 mL/min run time: 55 min temp: 25.degree. C. LC:
HP-1090 detection: 214, 280 nm injection: 15 .mu.g
[0096] Size Exclusion HPLC
5 column: Tosohaas TSK 2000SWXL, 5 .mu.m, 7.8 .times. 300 mm mobile
phase: 0.2 M potassium phosphate, 0.45 M KCl, pH 7.0 gradient:
isocratic flow rate: 1.0 mL/min run time: 30 min temp: ambient LC:
HP-1090 detection 214, 280 nm injection: 15 .mu.g
[0097] Cation Exchange HPLC
6 column: Tosohaas TSK SP-5PW, 10 .mu.m, 7.5 .times. 75 mm mobile
phase: A: 10 mM sodium phosphate, 10% (v/v) AcCN, pH 7.0 B: A + 1 M
ammonium chloride gradient: 10-40% B (60'), 40-60% B (5'), 60-10% B
(1'), 71-86% B (15') flow rate: 0.5 mL/min run time: 86 min temp:
35.degree. C. LC: HP-1090 detection 214 nm injection: 15 .mu.g
[0098] C. ELISA. This assay with a range of 0.39-6.25 ng/mL was
carried out by Immunoassay Services (Test Procedure Code SNGF:1 of
Genentech, Inc.). Each rhNGF sample was diluted in assay diluent to
two target concentrations of 5 and 2.5 ng/mL, and each dilution was
submitted in micronic tubes in triplicate. The protein
concentration in mg/mL was normalized to a -70.degree. C. internal
reference standard which was submitted for the same assay.
[0099] D. Radioreceptor Assay (RRA). This assay measures the
ability of unlabeled rhNGF to compete with .sup.125I-rhNGF for
receptor binding on PC-12 cells. This assay was carried out by
Bioassay Service (Genentech, Inc. Test Procedure SNGF:6) and has a
range of 3-80 ng/mL. Each rhNGF sample was diluted in assay diluent
to two target concentrators of 25 and 12.5 ng/mL, and each dilution
was submitted in micronic tubes in duplicate. The protein
concentration in mg/mL was normalized to a -70.degree. C. internal
reference standard which was submitted for the same assay.
[0100] E. PC-12 Cell Survival Bioassay. This assay determines the
ability of rhNGF to bind to its receptors and generate
intracellular signals that result in the survival of PC-12 cells
under serum-free culture conditions. This assay was carried out by
Bioassay Service (Test Procedure SNGF:7) and has a range of 0.24-30
ng/mL. The active protein concentration in mg/mL was normalized to
a -70.degree. C. internal reference standard which was submitted
for the same assay.
[0101] F. Visual Inspection. Visual inspection was performed on all
formulations in vials at the time of sampling. Samples were
observed for solution clarity, color, opalescence and particulate
formation.
[0102] G. pH Determination. pH of all formulations was determined
at each timepoint using a radiometer (model PHM82, Radiometer
America Inc.) and a micro-electrode (model M1-410, Microelectrodes,
Inc.). Standard solutions of pH 4.01 and pH 7.00 were used for the
standardization and calibration of the radiometer prior to pH
measurement.
[0103] H. Preservative Effectiveness Test. The lead rhNGF
multi-dose liquid formulations which were stable at 5.degree. C.
for 6 months were sent to Northview Lab for bacterial challenge
testing based on USP and EP standard criteria.
[0104] I. Circular Dichroism (CD) Analysis. An A VIV.RTM.
spectropolarimeter Model 60 DS equipped with water bath and data
processor was used to measure circular dichroism. Measurements were
made at 20.degree. C. Quartz cuvettes of 1.0 cm cell path length
was used for measuring near-UV CD. The CD spectra was taken at 0.2
nm intervals, with a 0.5 nm bandwidth, and 3.0 second averaging
time. Each sample for CD measurement was taken continuously for 24
hours. The CD data were expressed as the mean residue ellipticity
[q], degree.multidot.cm2/decimo- le, using the mean residue weight
of 120 for rhNGF.
[0105] Results
[0106] A preservative screening study was first performed to
examine the physical compatibility of several commonly used
preservatives with rhNGF at 0.1 mg/mL in the 20 mM sodium acetate
formulation at pH 5.5. These preservatives include benzyl alcohol,
phenol, m-cresol, methylparaben and propylparaben. In addition, the
physical compatibility of these preservatives with rhNGF in the
acetate formulation with the presence of surfactants such as
polysorbate 20 and pluronic acid (F68) was also studied. The
physical compatibility results are shown in Table 4.
7TABLE 4 List of rhNGF Liquid Formulations Selected for Long Term
Stability Testing I. Current liquid formulation 1. 2 mg/mL rhNGF in
10 mM acetate, 142 mM sodium chloride, pH 5.5 II. Control liquid
formulations (no preservative) 1. 0.1 mg/mL rhNGF in 20 mM acetate,
136 mM sodium chloride, pH 5.5 2. 0.1 mg/mL rhNGF in 20 mM acetate,
136 mM sodium chloride, 0.01% F68, pH 5.5 III. Multi-dose liquid
formulations 1. 0.1 mg/mL rhNGF in 20 mM acetate, 136 mM sodium
chloride, 0.9% benzyl alcohol, pH 5.5 2. 0.1 mg/mL rhNGF in 20 mM
acetate, 136 mM sodium chloride, 0.25% phenol, pH 5.5 3. 0.1 mg/mL
rhNGF in 20 mM acetate, 136 mM sodium chloride, 0.01% F68, 0.9%
benzyl alcohol, pH 5.5 4. 0.1 mg/mL rhNGF in 20 mM acetate, 136 mM
sodium chloride, 0.01% F68, 0.25% phenol, pH 5.5
[0107] Among the preservatives used for screening, they are all
physically compatible with rhNGF at 0.1 mg/mL in the acetate
formulation at pH 5.5. In the presence of polysorbate 20 at 0.01%
in the same formulation, only benzyl alcohol and phenol at final
concentrations of 0.9% and 0.25% respectively were physically
compatible with rhNGF. Phenol at 0.45% and m-cresol at 0.25% each
formed a cloudy solution with rhNGF in the acetate formulation in
the presence of polysorbate 20. The rhNGF solution also became
slightly opalescent upon the addition of methylparaben at 0.18% or
propylparaben at 0.02% to the polysorbate 20 containing acetate
formulation. On the other hand, pluronic acid at 0.01% in the same
formulation did not cause any physical incompatibility between
rhNGF and all the preservatives tested.
[0108] Based on the preservative screening study results, several
rhNGF, multi-dose liquid formulations containing either 0.9% benzyl
alcohol or 0.25% phenol in 20 mM acetate at pH 5.5 with and without
0.01% F68 were set up for long term stability study. A list of
these formulations were given in Table 5.
8TABLE 5 Physical Compatibility of Preservatives with 0.1 mg/mL
rhNGF Liquid Formulations Formulation buffer Surfactant
Preservative Results 20 mM acetate, none 0.9% benzyl alcohol co/cl
pH 5.5 0.25% phenol co/cl 136 mM NaCl 0.45% phenol co/cl 0.25%
m-cresol co/cl 0.18% methylparaben co/cl 0.02% propylparaben co/cl
20 mM acetate, Tween 20 0.9% benzyl alcohol co/cl pH 5.5 0.01%
0.25% phenol co/cl 136 mM NaCl 0.45% phenol cloudy 0.25% m-cresol
cloudy 0.18% methylparaben sl. opal 0.02% propylparaben sl. opal 20
mM acetate, 0.01% F68 0.9% benzyl alcohol co/cl pH 5.5 0.25% phenol
co/cl 136 mM NaCl 0.45% phenol co/cl 136 mM NaCl 0.25% m-cresol
co/cl 0.18% methylparaben co/cl 0.02% propylparaben co/cl
[0109] Stability of rhNGF in these formulations was assayed by the
following techniques: SE-HPLC, RP-HPLC, IE-HPLC, ELISA,
radioreceptor assay (RRA), PC-12 cell survival bioassay, pH, and
visual inspection. The acceptability of a multi-dose liquid
formulation for rhNGF will be based on comparison to the current
liquid formulation which consists of 2 mg/mL rhNGF in 10 mM sodium
acetate at pH 5.5, and 142 mM sodium chloride. In the other word,
the preserved formulation should be as stable as the current liquid
formulation. Results obtained to date represent 12 months at -70
and 5.degree. C., 9 months at 25.degree. C., and 3 months at
40.degree. C. stability monitoring data.
[0110] Size-Exclusion Chromatography. Size-exclusion HPLC was
employed to detect and quantitate aggregate formation in the rhNGF
multi-dose liquid formulations as well as their control
formulations which contain no preservative. Using this technique,
rhNGF elutes as dimer (main peak) at a retention time of 8.6
minutes. Benzyl alcohol and phenol elute at 16 and 19 minutes
respectively. The appearance of leading shoulder on the dimer main
peak indicates the presence of aggregate of higher molecular
weight. The data in Table 6 shows that rhNGF is stable to aggregate
formation in all formulations containing 0.9% benzyl alcohol as
preservative.
9TABLE 6 Effect of preservative on aggregation of 0.1 mg/mL rhNGF
in liquid formulations was determined by SEC-HPLC. Samples were
stored at 5.degree. C. for 12 months, 25.degree. C. for 9 months
and 40.degree. C. for 3 months. % Aggregate Formulation buffer
Surfactant Preservative 5.degree. C. 25.degree. C. 40.degree. C. 10
mM acetate, pH 5.5 none none 0 0.2 0.4 145 mM NaCl, 2 mg/mL 20 mM
acetate, pH 5.5 none none 0 0 0 136 mM NaCl, 0.1 mg/mL 20 mM
acetate, pH 5.5 none 0.9% benzyl. 0 0 0 136 mM NaCl, 0.1 mg/mL alc.
20 mM acetate, pH 5.5 none 0.25% phenol 0 0.4 0.5 136 mM NaCl, 0.1
mg/mL 20 mM acetate, pH 5.5 0.01% F68 none 0 0 0 136 mM NaCl, 0.1
mg/mL 20 mM acetate, pH 5.5 0.01% F68 0.9% benzyl 0 0 0 136 mM
NaCl, 0.1 mg/mL alc. 20 mM acetate, pH 5.5 0.01% F68 0.25% phenol 0
0.5 0.5 136 mM NaCl, 0.1 mg/mL
[0111] A small amount of aggregate (less than 1%) was detected in
the phenol containing formulations (with and without 0.01% F68 as
surfactant) after 3 months at 40.degree. C. and 9 months at
5.degree. C. Total protein recovery of these samples, compared to
their -70.degree. C. controls, was given in Table 7.
10TABLE 7 Quantitation of total rhNGF by SE-HPLC. Samples were
stored at 5.degree. C. for 12 months, 25.degree. C. for 9 months
and 40.degree. C. for 3 months. % Recovery Formulation buffer
Surfactant Preservative 5.degree. C. 25.degree. C. 40.degree. C. 10
mM acetate, pH 5.5 none none 102 102 102 145 mM NaCl, 2 mg/mL 20 mM
acetate, pH 5.5 none none 101 101 101 136 mM NaCl, 0.1 mg/mL 20 mM
acetate, pH 5.5 none 0.9% benzyl alc. 102 99 101 136 mM NaCl, 0.1
mg/mL 20 mM acetate, pH 5.5 none 0.25% phenol 99 97 98 136 mM NaCl,
0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68 none 101 101 98 136 mM
NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68 0.9% benzyl alc.
101 99 99 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68
0.25% phenol 100 97 97 136 mM NaCl, 0.1 mg/mL
[0112] Current formulation, controls and benzyl alcohol containing
formulations had 99% or greater protein recovery after 9 months at
25.degree. C., while phenol containing formulations had 97% for the
same storage time and temperature. These results indicate that
rhNGF is more compatible and stable with benzyl alcohol than phenol
in all formulations studied.
[0113] Reversed-Phase HPLC. The rhNGF used in this study consists
of mainly 118/118 homodimer and a small amount of 120/120
homodimer. Under the conditions of reversed-phase chromatography,
the two rhNGF dimeric forms are dissociated and their monomers are
separated. RP-HPLC separates the rhNGF monomers based on the
hydrophobicity of each species. The 118 monomer which is more
hydrophobic than the 120 monomer elutes at a retention time of 23
minutes. The 120 monomer elutes as a small peak in front of the 118
monomer peak. Comparison of RP-HPLC chromatograms of rhNGF in the
benzyl alcohol preserved formulation containing no surfactant at 5,
25, and 40.degree. C. are shown in FIG. 8. The degradation of rhNGF
stored at elevated temperatures was mainly due to the formation of
iso-aspartate, loss in 118 and 120 monomer peak areas, clip
formation and increase in misfolded rhNGF as determined by RP-HPLC.
The mono- and di-oxidized rhNGF peaks and the deamidated rhNGF peak
remain unchanged. In this study, rhNGF is defined as the sum of the
118 and 120 monomer peak areas by RP-HPLC, and the results are
reported as percent rhNGF remaining as compared to the -70.degree.
C. controls.
[0114] Decrease in percent protein remaining due to the loss of 118
and 120 monomer peak areas assayed by RP-HPLC is the major
degradation for rhNGF in liquid formulation. At 5.degree. C., the
stability of rhNGF in multi-dose formulations as determined by
RP-HPLC are essentially equivalent to the non-preserved control
formulations as well as the current formulation (more than 95%
rhNGF remaining after 12 months) except for the phenol preserved
formulation containing 0.01% F68 (FIG. 9). This formulation had
slightly less percent rhNGF remaining (93%) after 12 months at
5.degree. C. At 25.degree. C., rhNGF is obviously less stable in
the presence of 0.25% phenol than 0.9% benzyl alcohol as
preservative in the 20 mM acetate formulation at pH 5.5 (FIG. 10).
The combination of phenol and F68 in the acetate formulation caused
more degradation of the protein than the presence of phenol
alone.
[0115] Iso-aspartate formation of rhNGF in liquid form is time and
temperature dependent. The rate of iso-aspartate formation
increases with increase in time and temperature. At 5.degree. C.,
all formulations show a similar rate of iso-aspartate formation
(FIG. 11). There was about 1.5% iso-aspartate formed in all rhNGF
multi-dose formulations and their non-preserved control
formulations after 12 months at 5.degree. C. However, the rate of
iso-aspartate formation is slightly higher in the rhNGF
formulations preserved with 0.9% benzyl alcohol than the control
formulations and phenol preserved formulations stored at 25.degree.
C. (FIG. 12). Since iso-aspartate formation of rhNGF does not
affect the bioactivity of the protein, the effect of preservative
on iso-aspartate formation of rhNGF is not a major concern.
[0116] Cation Exchange Chromatography. IE-HPLC chromatograms for
rhNGF in the current formulation at 3 months at 5, 25, and
40.degree. C. are shown in FIG. 13. There are three major peaks
observed. The predominant peak is the 118/118 dimer (peak b) which
elutes at about 48 minutes. The peak c behind the main peak is from
a serine to glycine substitution at position 1 in one of the two
dimer chain. The peak a in front of the main peak is believed to be
the oxidized 118/118 and oxidized N-terminally clipped rhNGF. At
elevated temperatures (25 and 40.degree. C.), degradation of rhNGF
as determined by IE-HPLC is characterized by the decrease in peak
areas of the 118/118 main peak and the serine to glycine
substituted 118/118 dimer and the increase in peak a area. In this
study, rhNGF is defined as the sum of the 118/118 dimer (peak b)
and one chain serine to glycine dimer (peak c) peak areas by
IE-HPLC, and the results are reported as percent rhNGF remaining as
compared to the -70.degree. C. controls.
[0117] FIGS. 14 and 15 show the percent rhNGF remaining in all
rhNGF formulations by IE-HPLC after 12 months at 5.degree. C. and 9
months at 25.degree. C., respectively. At 5.degree. C., the peak
area of peaks b and c for all rhNGF formulations remained unchange
after 12 months. At 25.degree. C., all rhNGF formulations show a
similar rate of degradation, and there was no significant
difference in stability between the multi-dose formulations and the
control formulations as assessed by IE-HPLC.
[0118] ELISA. The data in Table 8 show the percent rhNGF remaining
at 5, 25 and 40.degree. C. after 12, 9 and 3 months of storage,
respectively.
11TABLE 8 Stability of current and selected multi-dose liquid
formulations for rhNGF determined by ELISA after 12 months at
5.degree. C., 9 months at 25.degree. C., and 3 months at 40.degree.
C. % rhNGF .sup.aRemaining Formulation buffer Surfactant
Preservative 5.degree. C. 25.degree. C. 40.degree. C. 10 mM
acetate, pH 5.5 none none 101.2 89.1 102.2 145 mM NaCl, 2 mg/mL 20
mM acetate, pH 5.5 none none 97.8 102.0 94.4 136 mM NaCl, 0.1 mg/mL
20 mM acetate, pH 5.5 none 0.9% benzyl 103.1 92.9 97.1 136 mM NaCl,
0.1 mg/mL alc. 20 mM acetate, pH 5.5 none 0.25% phenol 111.3 88.5
91.6 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68 none
98.5 102.7 92.7 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01%
F68 0.9% benzyl 101.9 92.6 87.7 136 mM NaCl, 0.1 mg/mL alc. 20 mM
acetate, pH 5.5 0.01% F68 0.25% phenol 103.4 92.5 82.6 136 mM NaCl,
0.1 mg/mL .sup.aCalculated as a percentage of assay response for
-70.degree. C. control sample at the same storage period.
[0119] Results were normalized to the -70.degree. C. controls
stored at the same temperature for the same period of time. There
were no significant difference between the benzyl alcohol and
phenol preserved formulation either in the presence or absence of
0.01% F68 as surfactant for all temperatures and time points
studied.
[0120] Radioreceptor Binding Activity (RRA). The RRA results are
presented in Table 9 and are normalized to the -70.degree. C.
controls.
12TABLE 9 Stability of current and selected multi-dose liquid
formulations for rhNGF determined by RRA after 12 months at
5.degree. C., 9 months at 25.degree. C., and 3 months at 40.degree.
C. % rhNGF .sup.aRemaining Formulation buffer Surfactant
Preservative 5.degree. C. 25.degree. C. 40.degree. C. 10 mM
acetate, pH 5.5 none none 111.3 121.5 74.9 145 mM NaCl, 2 mg/mL 20
mM acetate, pH 5.5 none none 100.6 106.5 82.1 136 mM NaCl, 0.1
mg/mL 20 mM acetate, pH 5.5 none 0.9% benzyl alc. 94.2 91.3 81.6
136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 none 0.25% phenol 82.0
72.5 68.8 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68
none 92.9 79.2 80.8 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5
0.01% F68 0.9% benzyl alc. 92.0 80.7 83.2 136 mM NaCl, 0.1 mg/mL 20
mM acetate, pH 5.5 0.01% F68 0.25% phenol 98.0 83.7 73.7 136 mM
NaCl, 0.1 mg/mL .sup.aCalculated as a percentage of assay response
for -70.degree. C. control sample at the same storage period.
[0121] In the absence of 0.01% F68 in the acetate formulation at pH
5.5, the phenol preserved formulation had less percent protein
remaining than both the benzyl alcohol preserved formulation and
the control formulation for all temperatures studied. In the
presence of 0.01% F68 in the acetate formulation at pH 5.5, rhNGF
in the preserved (benzyl alcohol or phenol) and the control
formulation had lost about 20% of its bioactivity at. 25 and
40.degree. C. after 9 and 3 months, respectively. These results
suggest that phenol and F68 can affect the ability of rhNGF to bind
to the NGF receptor on PC-12 cells. Therefore, benzyl alcohol at
0.9% is a better choice of preservative for rhNGF in the acetate
formulation containing no surfactant for multi-use purpose.
[0122] PC-12 Cell Survival Bioassay. In contrast to the RRA
results, the PC-12 cell survival bioassay data in Table 10 show
that there was no significant difference in potency of rhNGF in all
formulations stored at 5.degree. C. for 12 months and 25.degree. C.
for 9 months.
13TABLE 10 Stability of current and selected multi-dose liquid
formulations for rhNGF determined by bioassay after 12 months at
5.degree. C. and 9 months at 25.degree. C. % rhNGF .sup.aRemaining
Formulation buffer Surfactant Preservative 5.degree. C. 25.degree.
C. 10 mM acetate, pH 5.5 none none 101.7 96.1 145 mM NaCl, 2 mg/mL
20 mM acetate, pH 5.5 none none 84.3 113.7 136 mM NaCl, 0.1 mg/mL
20 mM acetate, pH 5.5 none 0.9% benzyl alc 102.2 97.3 136 mM NaCl,
0.1 mg/mL 20 mM acetate, pH 5.5 none 0.25% phenol 95.3 102.1 136 mM
NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68 none 101.3 95.9 136
mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68 0.9% benzyl alc.
96.6 94.2 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 0.01% F68
0.25% phenol 97.8 96.4 136 mM NaCl, 0.1 mg/mL .sup.aCalculated as a
percentage of assay response for -70.degree. C. control sample at
the same storage period.
[0123] The protein was found to be fully active in all formulations
as determined by this bioassay. Therefore, the radioreceptor
binding assay is a more stability indicating assay than the cell
survival bioassay in determining the bioactivity of rhNGF.
[0124] Solutions of all rhNGF formulations were clear and colorless
to the naked eyes (Table 11). Particulates were not observed in any
of the formulations at all temperatures and timepoints.
14TABLE 11 pH and visual clarity of rhNGF formulations after 12
months at 5.degree. C. and 9 months at 25.degree. C. Visual
Formulation pH Clarity pH Visual Clarity buffer 5.degree. C.
5.degree. C. 25.degree. C. 25.degree. C. 10 mM acetate, pH 5.5 5.50
co/cl 5.40 co/cl 145 mM NaCl, 2 mg/mL 20 mM acetate, pH 5.5 5.54
co/cl 5.41 co/cl 136 mM NaCl, 0.1 mg/mL 20 mM acetate, pH 5.5 5.52
co/cl 5.58 co/cl 136 mM NaCl, 0.1 mg/mL 0.9% benzyl alc. 20 mM
acetate, pH 5.5 5.49 co/cl 5.60 co/cl 136 mM NaCl, 0.1 mg/mL 0.25%
phenol 20 mM acetate, pH 5.5 5.47 co/cl 5.53 co/cl 136 mM NaCl, 0.1
mg/mL 0.01% F68 20 mM acetate, pH 5.5 5.42 co/cl 5.42 co/cl 136 mM
NaCl, 0.1 mg/mL 0.01% F68, 0.9% benzyl alc. 20 mM acetate, pH 5.5
5.48 co/cl 5.41 co/cl 136 mM NaCl, 0.1 mg/mL 0.01% F68, 0.25%
phenol co/cl = colorless and clear
[0125] pH Results. rhNGF formulated in 10 mM acetate, 142 mM sodium
chloride at either pH 5.0 or pH 5.8 had an increase in pH by 0.2
units during the stability study. The multi-dose formulations and
their control formulations used in this study were formulated in 20
mM acetate at pH 5.5 which should provide a higher buffer capacity
to prevent pH change. Table 11 shows that pH remained unchange for
all formulations studied.
[0126] Preservative Effectiveness Test. After 6 months of stability
study, the most stable multi-dose formulation for rhNGF which
consists of 0.1 mg/mL rhNGF in 20 mM acetate at pH 5.5, 136 mM
sodium chloride, and 0.9% benzyl alcohol was submitted for
preservative efficacy testing. This lead formulation passed both
the USP and EP (criteria A and B) after 6 months storage at
5.degree. C.
[0127] Circular Dichroism (CD) Analysis. The presence of 0.9%
benzyl alcohol in various liquid interferon-gamma (rhIFN-g)
formulations induces loss in circular dichroism signals in the
near-UV region. The near-UV CD signal of rhIFN-g disappeared within
24 hours, indicating that there was a change in tertiary structure
of the protein due to the presence of benzyl alcohol. However, this
phenomenon was not observed in the rhNGF formulation preserved with
0.9% benzyl alcohol. After 24 hours of the addition of the
preservative, the near-UV CD spectrum remained unchange, suggesting
that there is no interaction between rhNGF and benzyl alcohol in
the acetate formation at pH 5.5. FIG. 16 shows the near-UV CD
spectrum of rhNGF, and FIG. 17 compares the rear-UV CD spectra of
rhNGF in the presence and absence of benzyl alcohol after 24 hours
at 25.degree. C. Due to the interference of benzyl alcohol at
wavelength below 275 nm, CD spectrum of rhNGF was scanned from 325
nm to 275 nm when the sample contained the preservative.
[0128] Stresses Testing Stability
[0129] 1. Agitation Studies. Shaker studies were performed to
determine whether it is necessary to add surfactant (F68) in the
rhNGF multi-dose formulations at low protein concentration such as
0.1 mg/mL in order to prevent protein aggregation and maintain
visual clarity of the solutions during agitation. The Data of Table
12 show that rhNGF at 0.1 mg/mL in the 20 mM acetate formulation at
pH 5.5 (with or without preservative) is quite stable to mechanical
disruption such as shaking. This suggests that surfactant is not
required in formulating rhNGF at 0.1 mg/mL as multi-dose liquid
form for stability purpose.
15TABLE 12 Effect of agitation on stability of rhNGF multi-dose
liquid formulations. Samples were shaken at 80 rpm for 6 and 24
hours at room temperature. % Monomer % Iso-Asp % NGF ELISA RRA
Formulation Hours (SEC) (RP-HPLC) (RP-HPLC) (mg/mL) (mg/mL) 1 6 0
0.6 101.6 0.1 0.11 24 0.4 0.5 101.7 0.09 0.11 2 6 0 0.8 103.6 0.09
0.11 24 0 0.7 100.8 0.09 0.10 3 6 0 0.6 101.3 0.09 0.10 24 0 0.6
101.0 0.09 0.10 4 6 0 0.6 100.8 0.09 0.10 24 0 0.7 101.0 0.09 0.10
Formulations: 1. 2 mg/mL, 10 mM acetate pH 5.5, 145 mM NaCl. 2. 0.1
mg/mL, 20 mM acetate pH 5.5, 136 mM NaCl. 3. 0.1 mg/mL, 20 mM
acetate pH 5.5, 136 mM NaCl, 0.9% benzyl alcohol. 4. 0.1 mg/mL, 20
mM acetate pH 5.5, 136 mM NaCl, 0.25% phenol.
[0130] 2. Freezing-Thawing Studies. Results on the effect of
freezing and thawing on stability of 0.1 mg/mL rhNGF multi-dose
liquid formulations are presented in Table 13.
16TABLE 13 Effect of freeze-thaw on stability of rhNGF multi-dose
liquid formulations. Freeze -70.degree. C. % Aggregate % Iso-Asp %
NGF ELISA RRA Formulation Thaw 5.degree. C. (SEC) (RP-HPLC)
(RP-HPLC) (mg/mL) (mg/mL) 1 3 cycles 0 0.9 102.1 0.09 0.10 2 3
cycles 0 0.4 102.1 0.08 0.10 3 3 cycles 0 0.8 101.3 0.09 0.11 4 3
cycles 0 0.5 101.8 0.09 0.10 Formulations: 1. 2 mg/mL, 10 mM
acetate pH 5.5, 145 mM NaCl. 2. 0.1 mg/mL, 20 mM acetate pH 5.5,
136 mM NaCl. 3. 0.1 mg/mL, 20 mM acetate pH 5.5, 136 mM NaCl, 0.9%
benzyl alcohol. 4. 0.1 mg/mL, 20 mM acetate pH 5.5, 136 mM NaCl,
0.25% phenol.
[0131] After 3 cycles of freezing and thawing, the 0.1 mg/mL rhNGF
in the 20 mM acetate formulation at pH 5.5 as control and the two
multi-dose formulations containing either 0.9% benzyl alcohol or
0.25% phenol did not show any loss in stability of the protein.
They are as stable as the current 2 mg/mL rhNGF liquid formulation
after 3 cycles of freezing and thawing between -70 and 5.degree.
C.
[0132] 3. Light Compatibility Studies. Table 14 summarizes the
effect of light on stability of rhNGF in the current 2 mg/mL
formulation, the 0.1 mg/mL rhNGF control formulation, and the
benzyl alcohol or phenol preserved 0.1 mg/mL rhNGF
formulations.
17TABLE 14 Effect of light on stability of rhNGF multi-dose liquid
formulations. Samples were illuminated at a light intensity of
20,000 lux at 28.degree. C. Conc. Storage % Aggregate ELISA RRA
Formulation (mg/mL) Condition Weeks (SEC) (mg/mL) (mg/mL) 10 mM
acetate pH5.5 2 Dark 2 0 2.20 2.00 145 mM NaCl 5 0.3 2.20 2.00 20
mM acetate pH5.5 0.1 Dark 2 0 0.10 0.11 136 mM NaCl 5 0 0.09 0.10
20 mM acetate pH5.5 0.1 Dark 2 0 0.10 0.11 136 mM NaCl, 5 0 0.10
0.10 0.9% benzyl alcohol 20 mM acetate pH5.5 0.1 Dark 2 0 0.10 0.10
136 mM NaCl, 5 0.2 0.10 0.10 0.25% phenol 10 mM acetate pH5.5 2
Light 2 0.4 2.20 2.40 145 mM NaCl 5 1.6 2.00 1.80 20 mM acetate
pH5.5 0.1 Light 2 0 0.10 0.10 136 mM NaCl 5 0.3 0.09 0.09 20 mM
acetate pH5.5 0.1 Light 2 0 0.10 0.10 136 mM NaCl, 5 0.2 0.09 0.09
0.9% benzyl alcohol 20 mM acetate pH5.5 0.1 Light 2 0.7 0.09 0.10
136 mM NaCl, 5 12.1 0.07 0.04 0.25% phenol
[0133] After storage for 2 weeks in the light box, there was no
significant loss in stability of the protein in all formulations
studied. However, after 5 weeks of storage in the light box,
SE-HPLC indicated an increase in aggregate formation occurred in
the current formulation (1.6%). Aggregate formation was even more
pronounced in the phenol preserved formulation (12.1%) after 5
weeks exposure to light. There was also a 30% loss in protein
concentration and 60% in bioactivity in the light exposed phenol
containing formulation as determined by ELISA and RRA,
respectively. Both benzyl alcohol preserved formulation and the 0.1
mg/mL rhNGF control formulation were stable after exposure to light
for 5 weeks. All control vials wrapped with aluminum foil were
stable after 5 weeks of storage in the light box. These results
suggest that rhNGF is more sensitive to light at higher protein
concentration (2 mg/mL) than at lower protein concentration (0.1
mg/mL) in the acetate formulation at pH 5.5. In the presence of
phenol, rhNGF degrades more faster upon light exposure.
[0134] All 0.1 mg/mL rhNGF multi-dose liquid formation at pH 5.5
are stable at 5.degree. C. for 12 months. At 25.degree. C., the
formulations (with or without F68) using 0.25% phenol as
preservative were less stable than the formulations using 0.9%
benzyl alcohol. 0.1 mg/mL rhNGF Formulations at pH 5.5 containing
surfactant (F68) are as stable as the formulations containing no
surfactant.
[0135] The lead multi-dose formulation for rhNGF is 0.1 mg/mL
protein in 20 mM acetate, pH 5.5, 136 mM NaCl and 0.9% benzyl
alcohol filled in 3 cc vial with 1.8 mL filled. This formulation
passed both the USP and EP preservative efficacy testing after 6
month storage at 5.degree. C.
[0136] rhNGF at 0.1 mg/mL formulated in 20 mM acetate, 136 mM NaCl
pH 5.5 is as stable as the current 2 mg/mL liquid formulation.
[0137] Agitation has no effect on stability of rhNGF, with
regardless to protein concentration or excipient in the
formulation.
[0138] rhNGF is more stable in the dark than in the light
especially if the formulation contains phenol as preservative.
[0139] rhNGF at 2 mg/mL in the current formulation and at 0.1 mg/mL
in the multi-dose liquid formulations can undergo at least 3 cycles
of freezing (-70.degree. C.) and thawing (5.degree. C.) without any
adverse effect on the stability of the protein.
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