Note: Descriptions are shown in the official language in which they were submitted.
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PRESERVYIVG SECONDARY PEPTIDE STRUCTURE
Background of the Invention
Field Qf the Invention
[00011 The present invention is directed to a method of preserving the a-hetix
secondary
structure of certain peptides during freeze-drying, as well as to freeze-dried
formulations of
such peptides made according to the method.
Related Background Art
[0002) APPOI 8 and APL180 are known apolipoprotein (apo) A-I mimetics and are
disclosed in U.S. Patent Nos. 6,664,230 and 6,933,279 and WO 2004/034977,
respectively.
Each of these peptides comprises an 18 amino acid sequence, namely D-W-F-K-A-F-
Y-D-
K-V-A-E-K-F-K-E-A-F (Ac-Asp-Trp-Phe-Lys-Ala-Phe-Tyr-Asp-i.ys-Val-Ala-Glu-Lys-
Phe-Lys-Crlu-Ala-Phe-NH2 - SEQ II3 NO 1), having an acetyl amino-terminal
protecting
group and an amide carboxyl-terminal protecting group. In free (unbound) form,
when all
amino acids are in D-form, the peptide is known as APPOI 8; when all the amino
acids are in
L-form, the free (unbound) peptide is known as APLI 80. These peptides have
four
phenylalanines and are sometimes referred to as "D4F" when the amino acids are
all in the
D form or "IAF" when the amino acids are all in the L fonn. 12cverse "4F" is a
mirror
image of 4F with the relative positions of the amino acids to each other and
to the
hydrophilic and hydrophobic faces being identical. Similarly, peptides in this
group contain
two phenylalanines, known as 2F, three phenylalanines, known as 3F, five
phenylalanines,
5F, six phenylalanines, 6F and seven phenylalanines or 7F. It is possible to
have mirror
images or reverse peptides based on these peptides also.
(00031 All these peptides, have been shown to inhibit low density lipoprotein
(LDL)
oxidation, stimulate reverse cholesterol transport, and reduce formation of
atherosclerotic
lesion. Accordingly, these agents are useful in the treatment of
cardiovascular disease which
remains a leading cause of morbidity and mortality, particularly in the
llnited Statcs and in
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Western European countries. Hence, effective formulation of these peptides is
highly
desirable.
[0004] Exchangeable apolipoprotcins, including apo A-I, possess lipid-
associating domains
(Brouillette et al., Biochim. Biophys. Acta 1256:103-129 (1995); Segrest et
al., FEBS Lett.
38: _247-253 (1974)). Apo A-l has been postulated to possess eight tandem
repeating
22 mer sequences. Characteristics of the class A amphipathic helix include the
presence of
positively charged residues at the polar-nonpolar interface and negatively
charged residues
at the center of the polar face (Id.; Segrest et al., Proteins: Structure,
Function, and Genetics
8: 103-117 (1990)). Apo A-1 has been shown to strongly associate with
phospholipids to
form complexes and to promote cholesterol efflux from cholesterol-enriched
cells. It has
now been shown that the secondary structure of apo A-I is essential for high
affinity binding
to lipids, ultimately leading to its biological activity (Saito et alõ J.
Biol. Chem. 279(20):
20974-20981 (2004)). Hence, preservation of the secondary structure of apo A-I
is highly
desirable. Without limiting the invention to a particular mechanism of action,
it may be that
preservation of the a-helix conformation may be necessary for is important for
giving apo-I
its binding affinity to lipids.
10005] The invention of US Patent No. 6,664,230 which provided novel peptides
comprising 18 amino acids having a class A amphipathie helix when formulated
with "D"
amino acid residue(s) and/or having protected amino and carboxyl tennini which
when
orally administered to an organism, are readily taken up and delivered to the
serum, and are
effective to mitigate one or more symptoms of atherosclerosis.
100061 Freeze-drying proteins is a common approach to improve both chemical
and
physical stability of the proteirt. However, freezing and dehydration stress
can cause protein
aggregation, leading to a loss of it's bioactivity. Trehalose, a-D-
glucopyranosyl-a-D-
glucopyranoside, is a naturally occurring disaccharide, which has been shown
to be useful in
preventing denaturation of proteins and other macromolecules, viruses and
foodstuffs during
drying processes. See, e.g., U.S. Patent Nos_ 4,891,319, 5,149,653, 5,026,566,
5,902,565
and 6,890,512_ EP 0 762 897, while indicating that the method of preventing
aggregation
disclosed therein is applicable to both proteins and peptides, exemplifies its
method with
human growth hormone only. Trehalose has also been extensively studied as a
protein
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stabilizer in the literature (Kaushik et al., J. Bio. Chem. 278 (29): 26458-
26465 (2003)). To
date, no suggestion that trehalose may be effective in preserving peptide
secondary structure
has been noted in the prior art_
(00071 Accordingly, a method of preserving the a-helix (secondary) structure
of APP018
and APL180 during freeze-drying by trehalose would be desirable.
SummarY of the Invention
100061 The present invention is directed to a method of preserving secondary
structure
during freeze-drying of a peptide comprising the steps of: (a) admixing
trehalose with the
peptide in a solution, said trehalose in an amount sufficient to preserve
secondary strueture
of the peptide; and (b) freeze-drying the solution or suspension to obtain a
peptide
composition in which secondary structure has been prescrved, wherein the
peptide is
selected from N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-Lys-D-
Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phe-Amide (D4F); N-Acetyl-L-
Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-Lys-L-V a1-L-Ala-L-GIu-L-Lys-L-
Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide (L4F), D3F, L3F, DSF, L5F, D6F, L6F, D7F and
L7F or any phaxmaceutically acceptable salt form thereof. In a preferred
embodiment of this
aspect of the invention, the peptide is L4F. The invention is further directed
to a method
fiuther comprising the step of: (c) reconstituting the peptide composition to
obtain a
solution or suspension of the peptide in which secondary structure has been
preserved_
[0009] In certain preferred embodiments of the invention, the secondary
structure is an a-
helix structure. In other prefen:ed embodiments, the solution or suspension of
step (a)
further comprises at least one additional freeze-drying excipient such as
bufFer or surfactant.
100101 The present invention is further directed to freeze-dried and
reconstituted
compositions made according to the method of the invention.
[0011] The present invention is still further directed to a freeze-dried
composition
comprising a peptide and an amount of trehalose sufficient to preserve
secondary structure
of the peptide, wherein the peptide is selected from N-Acctyl-D-Asp-D-Trp-D-
Phe-D-Lys-
D-A la-D-Phe-D-Tyr-D-Asp-D-Lys-D- V al- D-Al a-D-GIu-D-Lys-D-Phe-D-Lys-D-Gl u-
D-
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Ala-D-Phe-Amide; N-Aceryl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-phe-L-Tyr-L-Asp-I.-
Lys-
L-Val-L-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-AIa-L-Phe-Amide, D3F, L3F, D5F,
L5F,
D6F, L6F, D7F and L7F or any pharmaceutically acceptable salt form thereof.
Detailed Description
100121 The present invention is directed to a method of preserving secondary
structure
during freeze-drying of a peptide_ As used herein, "secondary structure"
refers to the
general three-dimensional form of biomolecules such as peptides or of segments
of
biomolecules, such as proteins and nucleic aeids; for purposes of the present
invention,
"secondary structure" preferably refers to the a-helix structure of certain
peptides. As used
herein, "preserving" (and other forms thereof) refers to keeping intact.
Preserving
preferably refers to maintainence or improvement (increase) of the a-helix
content of certain
peptides - in other words, the a-helix content of a particular freeze-dried
composition made
according to the method of the present invention wil l be greater than that of
a freeze-dried
composition made according to conventional processes. As used herein, "freeze-
drying"
(and other forms thereof) refers to any process by which water is removed from
a material
whieh is first frozen and then subjected to reduced pressure and/or heat which
allows the
water to sublime directly from the solid phase to gas_
(0013j More specifically, the first embodiment of the presenl invention
comprises the steps
of: (a) admixing trehalose with the peptide in a solution or suspension, said
trehalose in an
amount sufficient to preserve secondary structure of the peptide; and (b)
freeze-drying the
solution or suspension to obtain a peptide composition in which secondary
structure bas
been preserved, wherein the peptide is selected from N-Acetyl-D-Asp-D-Trp-D-
Phe-D-Lys-
D-AIa-D-Phe-D-Tyr-D-Asp-D-Lys-D-Val-D-Ala-D-Glu-b-Lys-D-Phe-D-Lys-D-G1u-D-
AIa-D-Phe-Amide; N-Acetyl-C,-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-Phe-L-Tyr-L-Asp-L-
Lys-
L-Val-l,-Ala-L-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Araide, D3F, L3F, D5F,
L5F,
D6F, L6F, D7F and L7F or any pharmaceutically acceptable salt form thereof.
Preferably,
the peptide is the free form. N-Acetyl-I.-Asp-L-Trp-L-Phe-L-Lys-L-AIa-L-Phe-L-
Tyr-L:
Asp-L-Lys-L-Val-L-Ala-T.-Glu-L-Lys-L-Phe-L-Lys-L-Glu-L-AIa-L-Phe-Amide, The
individual peptides are generally referred to herein as a peptides of the
invention.
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(0014] In the first step of the inventive method, trehalose is admixed with a
peptide of the
invention in a solution.
(0015] Trehalose is a commercially available material and can be purchased
from any
source. Either the N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-Tyr-D-Asp-D-
I.ys-D-Val-D-Ala-D-Glu-D-Lys-D-Phe-D-Lys-D-Glu-D-Ala-D-Phe-Amide; N-Acetyl-L-
Asp-L-Trp-L-Phe-L-Lys-L-Ala-1.-Phe-L-Tyr-L-Asp-L-Lys-L-V al-L-AI a-L-Glu-L-Lys-
L-
Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide peptide, or any peptide of the invention,
can be
purchased from commercial sources or made according to known procedures as
described in
U.S. Patent Nos. 6,664,230 and 6,933,279 and PCT InternationaI Publication No.
WO 2004/034977, the entire disclosure of each of which is incorporated by
reference herein.
(0016] Trehalose and a peptide of the invention are adrnixed in water.
According to the
present invention, trehalose may be added to a solution of a peptide of the
invention, the
peptide may be added to a solution of trehalose or both trehalose and the
peptide may be
added to a solvent to form a solution in step (a). The pH is adjusted to a
range of from about
3 to 11, more preferably 6 to 9, even more preferably 6.5 to 9. Preferably a
surfactant,
including but not limited to TWBEN 80, is added prior to the addition of the
peptidc.
TWEEN 80 is present in an amount ranging preferably from about 0.0001 % to
about 10%
weight by volume.
(00171 The amount of trehalose suflicient to preserve secondary strueture of
the peptide
corresponds to a range preferably from about I to about 50%, more preferably
from about
to 25% weight by volume, with about 10% weight by volume being most preferred.
This
corresponds to a weight ratio of trehalose to peptide range of from about
500:0.01 to about
10:200, preferably of from about 250:0.2 to about 100:30 and most preferably
about 100:0.2
to about 100:30.
(0018] Admixing can be aecomplished by any conventional means, i.e., simple
mixture.
(0019] In a preferred embodiment of the present invention, the solution of
step (a) further
comprises at least one additional buffer. Buffers suitable for use in the
present invention
include, without limitation, sodium phosphate, for example mono or di sodium
phosphate,
potassium phosphate, Tris, citrate, tartrate and histidine and cornbinations
thereof. When
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present, phosphate buffer concent,ration corresponds to a range preferably
from about 1 mM
to about I M of the solution of step (a), preferably from about 5 mM to about
100 mM.
100201 In the second step of the inventive method, the solution or suspension
is freeze-dried
to obtain a peptide composition in which secondary structure has been
preserved. Freeze-
drying can be accomplished by any known means. For example, freeze-drying may
involve
the use of a freeze-drying flask which is rotated in a bath, which is cooled
by mechanical
refrigeration, dry ice and methanol, or liquid nitrogen or may involve the use
of a large-scale
freeze-drying machine. As a result of freeze-drying the combination of
trehalosc and
peptide, the secondary structure of the peptide in the peptide composition
will have been
preserved. In other words, the peptide composition of step (b) has a high a-
helix content as
compared to a peptide composition which was ffreeze-dried without the use of
trehalose.
[0021] An optional step for the first embodiment of the invention comprises
(c)
reconstituting the peptide composition to obtain a solution of the peptide in
which seoondary
structure has bcen preserved. Reconstitution can be accomplished by any known
means
such as by the simple addition of water to the peptide composition of step
(b). As one of
ordinary skill in the art will readily appreciate, solutions of varying
peptide concentration
can be achieved by reconstitution with varying amounts of solvent. Solvents
suitable for use
in step (c) include, without limitation, water, buffer solution or isotonic
solution. As a result
of the reconstitution of the freeze-dried peptide composition, the secondary
structure of the
peptide will have been preserved. In other words, the solution of step (c) has
a high peptide
secondary structure content, and possibly a high a-helix content as compared
to a solution or
suspension which was reconstituted from a freeze-dried composition which did
not use
trehalose in accordance with this invention.
[0022] Additional erabodiments of the invention are directed to freeze-dried
composition
and reconstituted compositions made according to the method of the first
embodiment of the
invention.
[0023] Yet another embodiment of the invention is directed to a freeze-dried
composition
comprising a peptide which is N-Acetyl-D-Asp-D-Trp-D-Phe-D-Lys-D-Ala-D-Phe-D-
Tyr-
D-Asp-D-Lys-D-V al -D-A1a-D-Glu-D-Lys-D-Phe-D-Lys-D-G lu-D-Aia-D-PhC- Amide;
N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-phe-L-Tyr-Y.-Asp-L-Lys-L-Val-L-Ala-L-
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Glu-L-Lys-l.-Phe-L-Lys-L-Glu-L-Ala-L-Phe-Amide, D3F, L3F, D5F, L5F, D6F, L6F,
D7F
and L7F or any pharmaceutically acceptable salt form thereof and an amount of
trehalose
sufficient to preserve the secondary structure of the peptide, Details
regarding the amounts
of peptide and trehalose are the same as those noted above with regard to the
firsl
embodiment of the invention.
100241 Specific embodiments of the invention will now be demonstrated by
reference to Ihe
following examples. It should be understood that these examples are disclosed
solely by
way of illustrating the invention and should not be taken in any way to limit
the scope of the
present invention.
EXAMPLE l
[0025] Freeze-dried compositions of N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-
Phe-L-
Tyr-L-Asp-L-Lys-L-Val-L-Ala-L-GIu-L-Lys-L-Phe-L-Lys-L-G1u-L-Ala-L-Phe-Amide
(APLI80) were made using the ingredients noted in Table I below.
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SYJSSTTTLTTE SHEET (RULE 26)
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~~g o 0
~
> I'i
~o..
Z' ry
bO
z N
N
`8 o O p
J, N o N p
Q C.
no
f.~
r-=
"" N N Q
C~,L
..i
SUBSTTTt)'TE SHEET (RULE 26)
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[0026] Formulations I and 3 were made for 1 mg/ml and formulations 2 and 4 for
100 mg/ml APL180. Both concentrations contain 15 mM phosphate buffer pH 7 and
10%
trehalose. Formulation 3 and 4 also contains 0.5% TWEEN 80. The solution of I
mg/ml
APL180 is prepared, filled at 1 ml per vial, freeze-dried, and reconstituted
with I ml water
prior to use. The formulation of 100 mg/ml APL180 is prepared at 25 mg/ml
APLISO
solution, filled at 2 ntl per via.l, freeze-dried, and reconstituted with 0.5
ml water prior to use.
Hence, other ingredients in the solution of25 mg/ml APL 180 are formulated at
25% of the
final concentration intended after reconstitution.
[0027) Lyophilization cycle is performed as follows:
Table 2.
Shelf
Step Operation ');'ime/[hh:mm] Temperature ( C) Chamber Pressure
1 Vial loading As required 20 Ambient
2 Freezing ramp 01:10 20 to -50 Ambient
3 Freezing hold Min, 03:00 -50 Ambient
Max. 70:00
4 Chamber vacuum 00:10 -50 0.111 mbar
Primary drying ramp 06:20 -50 to -12 0.111 mbar
6 Primary drying hold 24:00 -12 0.111 mbar
7 Secondary drying 06:10 -12 to 25 0.111 mbar
ramp
S Secondary drying 06:00 25 0.111 mbar
hold
Example 2 Freeze-Dried - Reconstituted Solution Study by Fourier Transform
Infrared Spectometry
(0028) Freeze-dried compositions of N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-
Phe-L-
Tyr-L-Asp-L-Lys-L-V aI-L-Ala-L-GIu-L-Lys-L-Phe-L-Lys-I.-GI u-L-AIa-L-Phe-Amide
(APLI 80) were made using the ingredients noted in Table 3 below and then
reconstituted as
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noted and tested using Fourier tru-sform infrared spectrometry to determine
the % a and %
(3 helices.
Table 3.
Formulation Concentration of
# Ingredient APL180 (mg/mi) % a % 0
1 1A-10% sucrose in phosphate 100 26 40
buffer
2 10 27 35
3 2A-10% trehalose in phosphate 100 29 38
buffer
4 10 43 28
5A-10% suorose + 0.5% 100 28 37
6 TWEEN 80 in phosphate buffer 10 28 31
7 6A-20% HPbCD in phosphate 100 26 38
8 buffer 10 24 32
9 7A-20% SBEbCD in phosphate 100 24 38
buffer 10 26 34
11 1 C-10% sucrose in tris buffer 100 22 42
12 10 20 39
13 1E-10% sucrose in histidine 100 23 41
14 buffer 10 n/a N/a
In phosphate buffer 100 n/a 42
16 10 n/a 34
17 In water 100 19 42
18 10 29 33
Example 3 Cotnpositions of 6 mg N-Acetyl-L-Asp-L-Trp-L-Phe-L-Lys-L-Ala-L-
Phe-L-Tyr-L-Asp-L-Lys-L-VaI-L-AIa-L-GIu-L-Lys-L-Phe-L-Lys-L-Glu-
L-Ala-L-Phe-Amide
100291 6 mg APL180 drug product is formulated as sterile, lyophilized powder
for
intravenous administration. The composition of each vial is provided in Table
4. Each vial
is overfilled with 2.2 ml of bulk solution before lyophilization and
reconstituted with 2 ml of
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water for injection (WFI) before administration. Two ml of reconstituted
solution will
deliver 6 mg APL 180.
Table 4. Composition of APY.180 drug product 6 mg/vi$l
Ingredients Amount per Vial Rationale for Use
Trehalose dihydrate 220.00 mg Lyo/Cryo-protectant
Disodium hydrogen phosphate 3_461 mg Buffering agent
anhydrous, USP/Ep
Natrium dihydrogen phosphate 1.342 mg Buffering agent
2AQ, U'SP/CP
Polysorbate 80, NF 0220 mg Surfactant
APLI 80 6_600 mg Active ingredient
= Primary packaging components
o vial drawn glass 6 ml/20mm blow back
o rubber stopper 20 mm Daikyo D777-1, V 10-F7-3 W B2-TR lyo, RS
o aluminum flip off PP/AL 20 mm nature/nature
. Bulk liquid formulation composition
ApLI80 is formulated as bulk liquid before being filled into vials and freeze-
dried, The
composition of the bulk liquid fonnulation is provided in Table 5.
Table S. Composition of API. 180 bulk liquid formulation before lyophilization
Ingredient Amount per ml
Trehalose dihydrate 100.00 mg
Disodium hydrogen phosphate anhydrous 1.573 mg
Natritun dihydrogen phosphate 2AQ 0.610 mg
Polysorbate 80 0.100 mg
APLl80 3.000 mg
Hydrocbloric acid 1.0 N As needed
Sodium hydroxide for injection As needed
WFI QS to 1.0 ml
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= Manufacturing procedures
Preparation of APL180 formulated bulk solution (Compounding)
1. Inspect compounding area and tank/vessel (SS T316L) for cleanliness.
2. Obtain tare weight of compounding vessel
3. Calculate the amount of WFI to add into compounding vessel (about 80% of
the total
final formulation volume).
4. Add the calculated amount of cool WFI to the compounding vessel_
5. Start a propeller mixer and adjust the speed to a moderate mixing. Add the
weighed
amounts of:
a. disodium hydrogen phosphate anhydrous
b_ nAtrium dihydrogen phosphate 2Aq.
c. trehalose dihydrate
d, polysorbate 80
APL180 will not be added until a, b, c, and d are completely dissolved. Mix
for at least
15 minutes. If not dissolved, continue mixing until dissolved by visual
inspection.
e. APL180
6. Add WF) to 98% of the full batch size. Pull a sample for pH. Adjust pH
using 1.0 N
sodium hydroxide for injection or 1 N HCL if neeessary.
7. QS to full batch size with WFI and mix for a minimum of five (5) minutes.
8. Take a sample for IPC testing for appearance, density and pH.
9. Test integrity of two 0.22 PVDF filters (Millipak 40 or Millipak 20
depending on
batch size) using the compounded solution. Discard all the solution used for
the integrity
testing.
Product specific BPmin = 38.5 psi
10, Filter the compounded solution through the integrity tested filters.
Discard the first
500 na) of the solution through the second filter.
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Y.yophiftation of AP'L180 formulation
11, Aseptically fill 2.2 ml of the bulk solution prepared from Step 10 into
clean and
sterile 6 ml vial. Discard the first 230 vials (equivalent to 506 ml of
solution).
12. Partially insett Daikyo D777-1 lyo rubber stoppers onto the vials.
13. Load the vials into a lyophilizer.
14. Start the lyophilization cycle by following the steps in Table 6_
15. At the end of the cycle, backfill the lyophilization chamber with nitrogen
gas with a
final chamber pressure of 850 50 mbar, Vials will then be fully stoppered.
16. Cap the inspected vials with Aluminum Flip Off seals_
Tab1e 6. Lyophilization Cycle Parameters
Time jhh:mm] Shelf Chambcr
Step Operation (rate) Temperature ( C) Pressure
I Vial loading As required Ambient Ambient
2 Cooling for As required 5 Ambient
uniformity (1 C/min)
3 Cooling hold 00:30 5 Ambient
4 Freezing ramp 01:30 (0.5 C/rtain) -40 Ambient
Freezing hold 03:00 -40 Ambient
6 Chamber vaeuuiri As required -40 0.10 mbar
7 Primary drying 00:22 (1 C/min) -18 0.10 mbar
ramp
8 Primary drying hold 32:00 -18 0.10 mbar
9 Secondary drying 01:26 (0.5 C/min) 25 0.10 mbar
ramp
Secondary drying 10:00 25 0.10 mbar
hold
11 Ending cycle 00:20 (1 C/rnin) 5 0.10 mbar
12 Preparing for As required 5 850 mbar
stoppering, nitrogen
back-filled
13 Shelf collapse As required 5 850 mbar
14 Hold for unload Min 00:00 5 850 mbar
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Max 24:00
[0030] While the invention has been described above with reference to specific
embodiments thereof, it is apparent that many changes, modifications, and
variations can be
made without departing from the inventive concept disclosed herein.
Accordingly, it is
intended to embrace all such changes, modifications, and variations that fall
within the spirit
and broad scope of the appended claims. All patent applications, patents, and
oEher
r publications cited herein are incorporated by reference in their entirety.
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