U.S. patent application number 14/116483 was filed with the patent office on 2014-03-27 for freeze-dried preparation containing high-purity pth and method for producing same.
This patent application is currently assigned to ASAHI KASEI PHARMA CORPORATION. The applicant listed for this patent is Takuji Maejima, Yoshiro Mitome, Fumihide Nishio. Invention is credited to Takuji Maejima, Yoshiro Mitome, Fumihide Nishio.
Application Number | 20140088014 14/116483 |
Document ID | / |
Family ID | 47296001 |
Filed Date | 2014-03-27 |
United States Patent
Application |
20140088014 |
Kind Code |
A1 |
Nishio; Fumihide ; et
al. |
March 27, 2014 |
FREEZE-DRIED PREPARATION CONTAINING HIGH-PURITY PTH AND METHOD FOR
PRODUCING SAME
Abstract
[Problem] Provided is a freeze-dried preparation containing
high-purity PTH peptide and a method for the production thereof.
Also provided is a test method for PTH analogs to confirm the
purity of a freeze-dried preparation containing PTH peptide, and
the like. [Solution] In the present invention, the presence of PTH
analogs produced during the manufacturing process of a freeze-dried
preparation containing PTH peptide was confirmed. The production of
these PTH analogs was also discovered to be markedly prevented or
reduced by controlling exposure of the solution containing PTH
peptide and the like to air environments within a pharmaceutical
production facility.
Inventors: |
Nishio; Fumihide; (Tokyo,
JP) ; Maejima; Takuji; (Tokyo, JP) ; Mitome;
Yoshiro; (Tokyo, JP) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Nishio; Fumihide
Maejima; Takuji
Mitome; Yoshiro |
Tokyo
Tokyo
Tokyo |
|
JP
JP
JP |
|
|
Assignee: |
ASAHI KASEI PHARMA
CORPORATION
Tokyo
JP
|
Family ID: |
47296001 |
Appl. No.: |
14/116483 |
Filed: |
May 31, 2012 |
PCT Filed: |
May 31, 2012 |
PCT NO: |
PCT/JP2012/064229 |
371 Date: |
November 8, 2013 |
Current U.S.
Class: |
514/11.8 |
Current CPC
Class: |
A61K 38/29 20130101;
A61P 43/00 20180101; C07K 14/635 20130101; G01N 30/7233 20130101;
A61P 5/18 20180101; A61P 19/10 20180101; A61P 5/20 20180101; B65B
63/08 20130101; B65B 55/12 20130101; B65B 55/10 20130101; A61K 9/19
20130101 |
Class at
Publication: |
514/11.8 |
International
Class: |
A61K 9/19 20060101
A61K009/19; A61K 38/29 20060101 A61K038/29 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 7, 2011 |
JP |
2011-127698 |
Claims
1. A freeze-dried preparation containing high-purity PTH peptide as
an active ingredient, wherein "high-purity" means at least that the
amount of at least one PTH analog versus the sum of the amount of
PTH peptide and the total amount of PTH analogs in the preparation
is 1.0% or less and/or that the total amount of PTH analogs versus
the sum of the amount of PTH peptide and the total amount of PTH
analogs is 5.0% or less; the freeze-dried preparation containing
PTH peptide being produced by a method characterized in that the
exposure of the solution containing PTH peptide prior to freeze
drying to air environments within a pharmaceutical production
facility is controlled.
2. The freeze-dried preparation containing PTH peptide set forth in
claim 1 wherein the PTH analog is at least one or more among 1)
analog 1: oxide of PTH peptide having a mass number 64 Da larger
than the mass number of the PTH peptide contained in the
preparation and producing digestion products corresponding to the
following fragments (1-a) to (1-c) when the analog is digested by
trypsin, (1-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da, (1-b) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID
NO: 2)+16 Da, and (1-c) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID
NO: 3)+4 Da; 2) analog 2: oxide of PTH peptide having a mass number
36 Da larger than the mass number of the PTH peptide contained in
the preparation and producing digestion products corresponding to
the following fragments (2-a) to (2-c) when the analog is digested
by trypsin, (2-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da, (2-b) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID
NO: 2)+16 Da, and (2-c) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID
NO: 3)+4 Da; 3) analog 3: oxide of PTH peptide having a mass number
32 Da larger than the mass number of the PTH peptide contained in
the preparation and producing digestion products corresponding to
the following fragments (3-a) and (3-b) when the analog is digested
by trypsin, (3-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da and (3-b) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ
ID NO: 2)+16 Da; 4) analog 4: oxide of PTH peptide having a mass
number 48 Da larger than the mass number of the PTH peptide
contained in the preparation and producing digestion products
corresponding to the following fragments (4-a) and (4-b) when the
analog is digested by trypsin, (4-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da, (4-b) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da; 5) analog 5: oxide of PTH peptide having a mass number 48 Da
larger than the mass number of the PTH peptide contained in the
preparation and producing digestion products corresponding to the
following fragments (5-a) and (5-b) when the analog is digested by
trypsin, (5-a) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID
NO: 2)+16 Da and (5-b) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID
NO: 3)+4 Da; 6) analog 6: oxide of PTH peptide having a mass number
20 Da larger than the mass number of the PTH peptide contained in
the preparation and producing digestion products corresponding to
the following fragments (6-a) and (6-b) when the analog is digested
by trypsin, (6-a) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ
ID NO: 2)+16 Da and (6-b) Mass number of Val-Glu-Trp-Leu-Arg (SEQ
ID NO: 3)+4 Da; 7) analog 7: oxide of PTH peptide having a mass
number 16 Da larger than the mass number of the PTH peptide
contained in the preparation and producing a digestion product
corresponding to the following fragment (7-a) when the analog is
digested by trypsin, (7-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da; 8) analog 8: oxide of PTH peptide having a mass number 16
Da larger than the mass number of the PTH peptide contained in the
preparation and producing a digestion product corresponding to the
following fragment (8-a) when the analog is digested by trypsin,
(8-a) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO: 2)+16
Da; 9) analog 9: oxide of PTH peptide having a mass number 32 Da
larger than the mass number of the PTH peptide contained in the
preparation and producing a digestion product corresponding to the
following fragment (9-a) when the analog is digested by trypsin,
(9-a) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4 Da; 10)
analog 10: oxide of PTH peptide having a mass number 16 Da larger
than the mass number of the PTH peptide contained in the
preparation and producing a digestion product corresponding to the
following fragment (10-a) when the analog is digested by trypsin,
(10-a) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+16 Da; or
11) analog 11: oxide of PTH peptide having a mass number 4 Da
larger than the mass number of the PTH peptide contained in the
preparation and producing a digestion product corresponding to the
following fragment (11-a) when the analog is digested by trypsin,
(11-a) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4 Da.
3. A freeze-dried preparation containing PTH peptide set forth in
claim 1 wherein the PTH analog is at least one or more among 1)
analog 1': oxide of PTH peptide in which residues corresponding to
the position 8 and position 18 methionine of human PTH (1-34) have
been changed into methionine sulfoxide residues and the residue
corresponding to the position 23 tryptophan has been changed into a
residue shown by the following structural formula (a); ##STR00007##
2) analog 2': oxide of PTH peptide in which residues corresponding
to the position 8 and position 18 methionine of human PTH (1-34)
have been changed into methionine sulfoxide residues and the
residue corresponding to the position 23 tryptophan has been
changed into a residue shown by the following structural formula
(b); ##STR00008## 3) analog 3': oxide of PTH peptide in which
residues corresponding to the position 8 and position 18 methionine
of human PTH (1-34) have been changed into methionine sulfoxide
residues; 4) analog 4': oxide of PTH peptide in which the residue
corresponding to the position 8 methionine of human PTH (1-34) has
been changed into a methionine sulfoxide residue and the residue
corresponding to the position 23 tryptophan has been changed into a
residue shown by the above structural formula (a); 5) analog 5':
oxide of PTH peptide in which the residue corresponding to the
position 18 methionine of human PTH (1-34) has been changed into a
methionine sulfoxide residue and the residue corresponding to the
position 23 tryptophan has been changed into a residue shown by the
above structural formula (a); 6) analog 6': oxide of PTH peptide in
which the residue corresponding to the position 18 methionine of
human PTH (1-34) has been changed into a methionine sulfoxide
residue and the residue corresponding to the position 23 tryptophan
has been changed into a residue shown by the above structural
formula (b); 7) analog 7': oxide of PTH peptide in which the
residue corresponding to the position 8 methionine of human PTH
(1-34) has been changed into a methionine sulfoxide residue; 8)
analog 8': oxide of PTH peptide in which the residue corresponding
to the position 18 methionine of human PTH (1-34) has been changed
into a methionine sulfoxide residue; 9) analog 9': oxide of PTH
peptide in which the residue corresponding to the position 23
tryptophan of human PTH (1-34) has been changed into a residue
shown by the above structural formula (a); 10) analog 10': oxide of
PTH peptide in which the residue corresponding to the position 23
tryptophan of human PTH (1-34) has been changed into a tryptophan
monoxide residue shown by the following structural formula (c-1) or
(c-2); ##STR00009## or 11) analog 11': oxide of PTH peptide in
which the residue corresponding to the position 23 tryptophan of
human PTH (1-34) has been changed into a residue shown by the above
structural formula (b).
4. The freeze-dried preparation containing PTH peptide set forth in
claim 2 wherein high-purity means that the amount of at least one
of the above analogs 1 to 11 versus the sum of the amount of PTH
peptide and the total amount of PTH analogs in the preparation is
1.0% or less and/or that the total amount of the above analogs 1 to
11 versus the sum of the amount of PTH peptide and the total amount
of PTH analogs is 5.0% or less.
5. The freeze-dried preparation containing PTH peptide set forth in
claim 3 wherein high-purity means that the amount of at least one
of the above analogs 1' to 11' versus the sum of the amount of PTH
peptide and the total amount of PTH analogs in the preparation is
1.0% or less and/or that the total amount of the above analogs 1'
to 11' versus the sum of the amount of PTH peptide and the total
amount of PTH analogs is 5.0% or less.
6. The freeze-dried preparation containing PTH peptide set forth in
claim 1 wherein the PTH peptide is human PTH (1-34).
7. The freeze-dried preparation containing PTH peptide set forth in
claim 1 wherein the freeze-dried preparation containing PTH peptide
is housed in a glass vial.
8. The freeze-dried preparation containing PTH peptide set forth in
claim 1, characterized in that exposure of the solution containing
PTH peptide to air environments within a pharmaceutical production
facility prior to freeze drying is controlled in any one or more
steps selected from a step for preparing a solution containing PTH
peptide, an aseptic filtration step, a drug solution dispensing
step, and a step for loading into a freeze drying means.
9. The freeze-dried preparation containing PTH peptide set forth in
claim 8 characterized in being produced using a method that also
includes control of exposure of the freeze-dried product to air
environments within a pharmaceutical production facility in a vial
sealing step after freeze drying.
10. The freeze-dried preparation containing PTH peptide set forth
in claim 1 characterized in that exposure of the solution
containing PTH peptide to air environments within a pharmaceutical
production facility prior to freeze drying is controlled in the
step for loading into the freeze drying means.
11. The freeze-dried preparation containing PTH peptide set forth
in claim 10 characterized in that the above exposure is controlled
by using a freeze-drying chamber equipped with means for
controlling the entrance of air within a pharmaceutical production
facility into the freeze-drying means.
12. The freeze-dried preparation containing PTH peptide set forth
in claim 11 characterized in that the freeze-drying means is a
freeze-drying chamber having an easily openable and closable
sub-door provided in an opening created in a small door unit opened
when containers housing the solution containing PTH peptide prior
to freeze drying are loaded into and unloaded from this means,
thereby controlling exposure of the solution containing PTH peptide
to air environments within a pharmaceutical production facility
prior to freeze drying by opening this sub-door only during
container loading and quickly closing the sub-door after
loading.
13. The freeze-dried preparation containing PTH peptide set forth
in claim 11 wherein the freeze-drying means is a freeze-drying
chamber having an opening created in a small door unit opened when
containers housing the solution containing PTH peptide prior to
freeze drying are loaded into and unloaded from this means, and the
means for controlling the ingress of air within a pharmaceutical
production facility into the freeze-drying means is an
airflow-adjusting cover that can change the air flow to a direction
not directed from this opening to the inside of the chamber.
14. The freeze-dried preparation containing PTH peptide set forth
in claim 10 characterized in that the loading step controls
exposure of the solution containing PTH peptide to air environments
within a pharmaceutical production facility prior to freeze drying
by purging the inside of the freeze-drying means with an inert
gas.
15. The freeze-dried preparation containing PTH peptide set forth
in claim 10 wherein the freeze-drying means is a freeze-drying
chamber having an easily openable and closable sub-door provided in
an opening created in a small door unit opened when containers
housing the solution containing PTH peptide prior to freeze drying
are loaded into and unloaded from this means, thereby controlling
exposure of the solution containing PTH peptide to air environments
within a pharmaceutical production facility prior to freeze drying
by opening this sub-door only during container loading and quickly
closing the sub-door after loading and purging the inside of the
freeze-drying means with an inert gas in the loading step.
16. The freeze-dried preparation containing PTH peptide set forth
in claim 10 wherein the freeze-drying means is a freeze-drying
chamber having an opening created in a small door unit opened when
containers housing the solution containing PTH peptide prior to
freeze drying are loaded into and unloaded from this means, this
opening being equipped with an airflow-adjusting cover, thereby
controlling exposure of the solution containing PTH peptide to air
environments within a pharmaceutical production facility prior to
freeze drying by changing the direction of the airflow-adjusting
cover so that air is not directed into the chamber and purging the
inside of the freeze-drying means with an inert gas in the loading
step.
17. The freeze-dried preparation containing PTH peptide set forth
in claim 10 wherein the loading step is a step that spans three or
more hours.
18. The freeze-dried preparation containing PTH peptide set forth
in claim 8 wherein time from the beginning of the step for
preparing a solution containing PTH peptide to the end of the step
for loading into the freeze-drying means spans three or more hours,
and production is performed using a method for controlling exposure
of the solution containing PTH peptide to air environments within a
pharmaceutical production facility in one or more steps during the
time.
19. The freeze-dried preparation containing PTH peptide set forth
in claim 14 wherein the inert gas is nitrogen gas.
20. A freeze-dried preparation containing high-purity PTH peptide
as an active ingredient, the PTH-peptide-containing freeze-dried
preparation manufactured using a method characterized in that
exposure of a solution containing PTH peptide to air environments
within a pharmaceutical production facility prior to freeze drying
is controlled during loading into the freeze-drying means; wherein
"high-purity" means at least that the amount of at least one PTH
analog versus the sum of the amount of PTH peptide and the total
amount of PTH analogs in the preparation is 1.0% or less and/or
that the total amount of PTH analogs versus the sum of the amount
of PTH peptide and the total amount of PTH analogs is 5.0% or less;
the loading step is a step that spans three or more hours; the air
environment is an environment that maintains one-way air flow of
clean air that has passed through an HEPA filter downward from
above; and the velocity of the air flow 20 cm directly under the
HEPA filter is 0.2-1.0 m/s.
21. A method for producing a freeze-dried preparation containing
PTH peptide, the method being characterized in that exposure of the
solution containing PTH peptide to air environments within a
pharmaceutical production facility is controlled in one or more
steps from the beginning of the step for preparing a solution
containing PTH peptide to the end of the step for loading into the
freeze-drying means.
22. The method set forth in claim 21 wherein exposure of the
freeze-dried product to air environments within a pharmaceutical
production facility is also controlled in the step for sealing
vials after freeze drying.
23. The method set forth in claim 21 characterized in that exposure
of the solution containing PTH peptide to air environments within a
pharmaceutical production facility is controlled in the step for
loading into the freeze-drying means.
24. The method set forth in claim 23 characterized in that exposure
is controlled using a freeze-drying chamber equipped with means for
controlling the inflow of air within a pharmaceutical production
facility into the freeze-drying means.
25. The method set forth in claim 23 wherein the step for loading
into the freeze-drying means is a step that spans three or more
hours.
26. The method set forth in claim 24 wherein the freeze-drying
means is a freeze-drying chamber having an easily openable and
closable sub-door provided in an opening created in a small door
unit opened when containers housing the solution containing PTH
peptide prior to freeze drying are loaded into and unloaded from
this means, thereby controlling exposure of the solution containing
PTH peptide to air environments within a pharmaceutical production
facility prior to freeze drying by opening this sub-door only
during container loading and quickly closing the sub-door after
loading.
27. The method set forth in claim 24 wherein the freeze-drying
means is a freeze-drying chamber having an opening created in a
small door unit opened when containers housing the solution
containing PTH peptide prior to freeze drying are loaded into and
unloaded from this means, and the means for controlling the ingress
of air within a pharmaceutical production facility into the
freeze-drying means is an airflow-adjusting cover that can change
the air flow to a direction not directed from this opening to the
inside of the chamber.
28. The method set forth in claim 23 characterized in that exposure
of the solution containing PTH peptide to air environments within a
pharmaceutical production facility prior to freeze drying is
controlled by purging the inside of the freeze-drying means with an
inert gas.
29. The method set forth in claim 23 characterized in that the
freeze-drying means is a freeze-drying chamber having an easily
openable and closable sub-door provided in an opening created in a
small door unit opened when containers housing the solution
containing PTH peptide prior to freeze drying are loaded and
unloaded, thereby controlling exposure of the solution containing
PTH peptide to air environments within a pharmaceutical production
facility prior to freeze drying by opening this sub-door only
during container loading and quickly closing the sub-door after
loading and purging the inside of the freeze-drying means with an
inert gas in the loading step.
30. The method set forth in claim 23, characterized in that the
freeze-drying means is a freeze-drying chamber having an opening
created in a small door unit opened when containers housing the
solution containing PTH peptide prior to freeze drying are loaded
into and unloaded from this means, the opening being equipped with
an airflow-adjusting cover, thereby controlling exposure of the
solution containing PTH peptide to air environments within a
pharmaceutical production facility prior to freeze drying by
changing the airflow-adjusting cover to a direction in which the
air flow is not directed into the chamber and purging the inside of
the freeze-drying means with an inert gas in the loading step.
31. The method set forth in claim 28 wherein the inert gas is
nitrogen.
32. The method set forth in claim 21 wherein the container housing
the solution containing PTH peptide is a glass vial.
33. The method set forth in claim 21 wherein the PTH is human PTH
(1-34).
34. The method set forth in claim 21 wherein the air environment
within a pharmaceutical production facility is an air environment
in which 1) the air is of grade A, 2) clean air that has passed
through an HEPA filter having the ability to trap particles having
a particle size of 0.3 .mu.m at an efficiency of 99.97% or higher
is maintained as a one-way air flow downward from above, and 3) the
ozone concentration is 0.001-0.1 ppm.
35. The method set forth in claim 21 wherein the air environment
within a pharmaceutical production facility is an air environment
containing a formaldehyde concentration of 0.1 ppm or less.
36. The method set forth in claim 21 wherein the amount of at least
one PTH analog versus the sum of the amount of PTH peptide and
total amount of PTH analogs is 1.0% or less and/or the total amount
of PTH analogs versus the sum of the amount of PTH peptide and
total amount of PTH analogs is 5.0% or less in the freeze-dried
preparation containing PTH peptide.
37. A method for producing a freeze-dried preparation containing
PTH peptide, the method being characterized in that exposure of the
solution containing PTH peptide to air environments within a
pharmaceutical production facility is controlled in one or more
steps from the beginning of the step for preparing a solution
containing PTH peptide to the end of the step for loading into the
freeze-drying means, for controlling the production of PTH analogs
1 to 11 set forth in claim 2.
38. A method for producing a freeze-dried preparation containing
PTH peptide, the method being characterized in that exposure of the
solution containing PTH peptide to air environments within a
pharmaceutical production facility is controlled in one or more
steps from the beginning of the step for preparing a solution
containing PTH peptide to the end of the step for loading into the
freeze-drying means, to control the production of PTH analogs to 1'
11' set forth in claim 3.
39. A freeze-dried preparation containing PTH peptide manufactured
using the method set forth in claim 21.
40. A method for producing a freeze-dried preparation containing
high-purity PTH peptide as an active ingredient, the method
characterized in that exposure of a solution containing PTH peptide
to air environments within a pharmaceutical production facility
prior to freeze drying is controlled during loading into the
freeze-drying means; wherein "high-purity" means at least that the
amount of at least one PTH analog versus the sum of the amount of
PTH peptide and the total amount of PTH analogs is 1.0% or less
and/or that the total amount of PTH analogs versus the sum of the
amount of PTH peptide and the total amount of PTH analogs is 5.0%
or less in the preparation; the loading step is a step that spans
three or more hours; the air environment is an environment in which
clean air that has passed through an HEPA filter is maintained as a
one-way air flow downward from above; and the velocity of the air
flow 20 cm directly under the HEPA filter is 0.2-1.0 m/s.
41. A method for testing a freeze-dried preparation containing PTH
peptide, the method being characterized in confirming the presence
of at least one or more of the PTH analogs 1 to 11 of claim 2
and/or determining the amounts present in the freeze-dried
preparation containing PTH peptide.
42. A method for testing a freeze-dried preparation containing PTH
peptide, the method being characterized in confirming the presence
of at least one or more of the PTH analogs 1' to 11' of claim 3
and/or determining the amounts present in the freeze-dried
preparation containing PTH peptide.
43. The method set forth in claim 41 wherein determination of the
PTH analogs includes calculating the area of the peak corresponding
to the PTH analog on a chromatogram when the ultraviolet absorption
of a sample derived from a freeze-dried preparation containing PTH
peptide is measured by high-performance liquid chromatography.
44. The method set forth in claim 43 including calculation of the
purity of the PTH peptide in the freeze-dried preparation
containing PTH peptide by comparing the area of a peak
corresponding to a PTH analog on a chromatogram and the peak area
corresponding to PTH peptide or the sum of the peak area of PTH
peptide and the peak area of all other PTH analogs detected on the
same chromatogram when the ultraviolet absorbance of a sample
derived from a freeze-dried preparation containing PTH peptide is
measured by high-performance liquid chromatography.
45. The method set forth in claim 44 including calculation of the
purity of the PTH peptide in a freeze-dried preparation containing
PTH peptide by comparing the area of that peak and the peak area
corresponding to PTH peptide or the sum of the peak area of PTH
peptide and the peak area of all other PTH analogs detected on the
same chromatogram when using chromatography conditions such that
any two or more PTH analogs are detected as one or more single
peaks on the chromatogram.
46. The method set forth in claim 41 for ensuring that the amount
of at least one PTH analog versus the sum of the amount of PTH
peptide and the total amount of PTH analogs is 1.0% or less and/or
the total amount of PTH analogs versus the sum of the amount of PTH
peptide and the total amount of PTH analogs is 5.0% or less in a
freeze-dried preparation containing PTH peptide.
47. The method set forth in claim 41 including the detection of the
mass number of the PTH analogs using a high-performance liquid
chromatograph-mass spectrometer.
48. The method set forth in claim 41 including fractionating a
substance that gives a single peak on the chromatogram and
identifying the mass number of the fragments produced by digesting
this substance using trypsin.
49. A method for producing a pharmaceutical comprising a
freeze-dried preparation containing PTH peptide including a step to
carry out the test method of claim 41.
Description
TECHNICAL FIELD
[0001] The present invention relates to a freeze-dried preparation
containing PTH (parathyroid hormone) or a substance of equivalent
physiologic activity (collectively referred to hereinafter as "PTH
peptide") as an active ingredient. The present invention also
relates to a method for producing a freeze-dried preparation
containing PTH peptide. The present invention also relates to a
method for testing and assuring the quality of freeze-dried
preparations containing PTH peptide.
BACKGROUND ART
[0002] Parathyroid hormone, together with calcitonins and vitamin
Ds, is a hormone that participates in regulating the calcium
concentration in the blood. Therefore, PTH peptide is used as a
diagnostic for hypoparathyroidism. Parathyroid hormone is also
known to accelerate the absorption of calcium in the intestine by
increasing active vitamin D3 production in the kidneys (Non-patent
Reference 1). A method of treating osteoporosis that increases the
density of cancellous bone and does not decrease the density of the
cortical bone of osteoporosis patients by subcutaneous
administration of 100 or 200 units/time of PTH once a week over a
period of 26 weeks to osteoporosis patients has also been disclosed
(Patent Reference 7).
[0003] A method of combining mannitol or another such saccharide or
gelatin or another such macromolecular substance as a stabilizer is
generally used when making a trace of PTH peptide into a
freeze-dried preparation to be dissolved at the time of use (Patent
References 1 and 2). A freeze-dried pharmaceutical composition
characterized in containing a monosaccharide or disaccharide and
sodium chloride is also known (Patent Reference 3).
[0004] When a freeze-dried preparation such as the above is
manufactured aseptically to produce a pharmaceutical, ordinary
pharmaceutical production facilities utilize areas that achieve a
sterile environment by a stream of aseptic air of a constant speed
that has passed through HEPA filters. A manufacturing process in a
pharmaceutical production facility under this sterile environment
typically consists of a step for preparing an active ingredient
solution, followed by a step for aseptically filtering of the
solution and dispensing it into containers, a step for loading the
filled containers into a freeze-drying chamber, and a step for
sealing the containers (vials and the like) after the freeze-drying
step.
PRIOR ART REFERENCES
Patent References
[0005] Patent Reference 1: JP Kokai 63-60904 [0006] Patent
Reference 2: JP Kokai 2-111 [0007] Patent Reference 3: JP Kokai
5-306235 [0008] Patent Reference 4: JP Kokai 64-16799 [0009] Patent
Reference 5: WO02/002136 [0010] Patent Reference 6: JP Kokai
2003-095974 [0011] Patent Reference 7: JP Kokai 8-73376 [0012]
Patent Reference 8: WO00/10596 [0013] Patent Reference 9:
WO10/30670
Non-Patent References
[0013] [0014] Non-Patent Reference 1: Current Osteoporosis Reports,
Vol. 6, 12-16, 2008 [0015] Non-Patent Reference 2: Journal of
pharmaceutical sciences, vol. 98, no. 12, p 4485-4500, 2009 [0016]
Non-Patent Reference 3: ADVANCES IN ENZYMOLOGY, 32, 221-296, 1969
[0017] Non-Patent Reference 4: J. Biol. Chem., vol. 266, 2831-2835,
1991 [0018] Non-Patent Reference 5: M. Takei et al., Peptide
Chemistry 1980, 187-192, 1981
SUMMARY OF INVENTION
Problems to be Solved by the Invention
[0019] The active ingredient of a pharmaceutical is obtained by
chemical synthesis from raw materials, by isolation and refining of
a biological product, by production by genetic engineering and
isolation and refining of the product, and the like. It is
generally difficult to obtain 100% purity of the pharmaceutical
active ingredient produced due to the purity of the raw materials
themselves, incomplete reactions, decomposition during isolation
and refining, and other such factors, in any method, including
genetic recombination. On the other hand, since the possibility of
undesirable effects on diagnosis and treatment cannot be ruled out
when diagnostic and therapeutic drugs contain more than the
acceptable amount of impurities, the fact remains that obtaining a
high-purity product is an important factor for producing a safe,
effective drug. When preparations containing PTH peptide are used
in the treatment/prevention of osteoporosis in particular, high
purity can be said to be especially necessary for preparations
containing PTH peptide because the duration of administration
extends over a long period of time.
[0020] However, it was found that, when the freeze-dried
preparation containing PTH peptide of the present invention is
manufactured on an industrial scale by a typical production process
such as the above, a preparation containing substances in which the
chemical structure of the active ingredient (PTH peptide) has been
changed (referred to hereinafter as "PTH analogs") is produced.
Especially as the production scale increases, the problem faced is
that there is concern that the amount of PTH analogs produced will
rise to an essentially unacceptable level as the production volume
increases. Furthermore, the amount of PTH analogs produced is not
always constant but changes depending the production time and
place, on differences in time, and the like. Since the factors that
lead to the production of these PTH analogs had not been specified,
a serious problem faced in practice was also that the amounts
produced could not be controlled.
[0021] The purpose of the present invention is to provide a
freeze-dried preparation containing PTH peptide of high purity,
that is, in which the content of PTH analogs is kept to an
acceptably low level. Another purpose of the present invention is
to provide a method for producing this high-purity freeze-dried
preparation containing PTH peptide. Yet another purpose of the
present invention is to provide a test method for PTH analogs for
purposes such as checking the purity of a freeze-dried preparation
containing PTH peptide.
Means Used to Solve the Above-Mentioned Problems
[0022] The present inventors were concerned that the amount of PTH
analogs produced would rise to an essentially unacceptable level as
the production scale increased and the production volume rose and
succeeded in isolating and characterizing these PTH analogs. They
also discovered that controlling the exposure of solution
containing PTH peptide and the like to air environments within a
pharmaceutical production facility greatly inhibits and decreases
the production of these PTH analogs.
[0023] While not being bound by the theory, it was assumed that,
given the structural characteristics of the PTH analogs
characterized as mentioned above and the fact that the production
of these analogs is inhibited and decreased by controlling exposure
to air environments within a pharmaceutical production facility,
the cause of the production of these PTH analogs is substances
having oxidizing capability present in air environments within a
pharmaceutical production facility. Certainly, aside from those of
high cleanliness (grade A and the like), air environments in
pharmaceutical production facilities often can contain gaseous
substances having oxidizing capability. Namely, pharmaceutical
production facilities are fumigated and disinfected by
formaldehyde, ozone, and other such sterilizing agents to more
fully actualize an aseptic environment. One can therefore also come
to think that gases having oxidizing capability such as
formaldehyde and ozone can be contained as residues of this
fumigation and disinfection. For that matter, ozone is present in a
concentration of 0.001-0.02 ppm, approximately 0.02-0.1 ppm
depending on the time, location, and season, in the atmosphere
regardless of fumigation and disinfection.
[0024] The present inventors also confirmed that the production of
the PTH analogs elucidated by the present invention can be
reproduced by bringing PTH peptide into contact with air containing
ozone.
[0025] The present invention therefore encompasses the following
aspects and preferred embodiments.
[0026] [1] A freeze-dried preparation containing high-purity PTH
peptide as an active ingredient, wherein "high-purity" means at
least that the amount of at least one PTH analog versus the sum of
the amount of PTH peptide and the total amount of PTH analogs in
the preparation is 1.0% or less and/or that the total amount of PTH
analogs versus the sum of the amount of PTH peptide and the total
amount of PTH analogs is 5.0% or less; the freeze-dried preparation
containing PTH peptide being produced by a method characterized in
that the exposure of the solution containing PTH peptide prior to
freeze drying to air environments within a pharmaceutical
production facility is controlled.
[0027] [2] A freeze-dried preparation containing PTH peptide set
forth in [1] wherein the PTH analog is at least one or more
among
[0028] 1) analog 1:
[0029] oxide of PTH peptide having a mass number 64 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing digestion products corresponding to the following
fragments (1-a) to (1-c) when the analog is digested by
trypsin,
[0030] (1-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da,
[0031] (1-b) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO:
2)+16 Da, and
[0032] (1-c) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da;
[0033] 2) analog 2:
[0034] oxide of PTH peptide having a mass number 36 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing digestion products corresponding to the following
fragments (2-a) to (2-c) when the analog is digested by
trypsin,
[0035] (2-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da,
[0036] (2-b) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO:
2)+16 Da, and
[0037] (2-c) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da;
[0038] 3) analog 3:
[0039] oxide of PTH peptide having a mass number 32 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing digestion products corresponding to the following
fragments (3-a) and (3-b) when the analog is digested by
trypsin,
[0040] (3-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da,
[0041] (3-b) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO:
2)+16 Da;
[0042] 4) analog 4:
[0043] oxide of PTH peptide having a mass number 48 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing digestion products corresponding to the following
fragments (4-a) and (4-b) when the analog is digested by
trypsin,
[0044] (4-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da,
[0045] (4-b) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da;
[0046] 5) analog 5:
[0047] oxide of PTH peptide having a mass number 48 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing digestion products corresponding to the following
fragments (5-a) and (5-b) when the analog is digested by
trypsin,
[0048] (5-a) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO:
2)+16 Da, and
[0049] (5-b) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da;
[0050] 6) analog 6:
[0051] oxide of PTH peptide having a mass number 20 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing digestion products corresponding to the following
fragments (6-a) and (6-b) when the analog is digested by
trypsin,
[0052] (6-a) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO:
2)+16 Da, and
[0053] (6-b) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da;
[0054] 7) analog 7:
[0055] oxide of PTH peptide having a mass number 16 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing a digestion product corresponding to the following
fragment (7-a) when the analog is digested by trypsin,
[0056] (7-a) Mass number of
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys (SEQ ID NO:
1)+16 Da;
[0057] 8) analog 8:
[0058] oxide of PTH peptide having a mass number 16 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing a digestion product corresponding to the following
fragment (8-a) when the analog is digested by trypsin,
[0059] (8-a) Mass number of His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO:
2)+16 Da;
[0060] 9) analog 9:
[0061] oxide of PTH peptide having a mass number 32 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing a digestion product corresponding to the following
fragment (9-a) when the analog is digested by trypsin,
[0062] (9-a) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da;
[0063] 10) analog 10:
[0064] oxide of PTH peptide having a mass number 16 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing a digestion product corresponding to the following
fragment (10-a) when the analog is digested by trypsin,
[0065] (10-a) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+16
Da; or
[0066] 11) analog 11:
[0067] oxide of PTH peptide having a mass number 4 Da larger than
the mass number of the PTH peptide contained in the preparation and
producing a digestion product corresponding to the following
fragment (11-a) when the analog is digested by trypsin,
[0068] (11-a) Mass number of Val-Glu-Trp-Leu-Arg (SEQ ID NO: 3)+4
Da.
[0069] [3] A freeze-dried preparation containing PTH peptide set
forth in [1] wherein the PTH analog is at least one or more
among
[0070] 1) analog 1':
[0071] oxide of PTH peptide in which residues corresponding to the
position 8 and position 18 methionine of human PTH (1-34) have been
changed into methionine sulfoxide residues and the residue
corresponding to the position 23 tryptophan has been changed into a
residue shown by the following structural formula (a);
##STR00001##
[0072] 2) analog 2':
[0073] oxide of PTH peptide in which residues corresponding to the
position 8 and position 18 methionine of human PTH (1-34) have been
changed into methionine sulfoxide residues and the residue
corresponding to the position 23 tryptophan has been changed into a
residue shown by the following structural formula (b);
##STR00002##
[0074] 3) analog 3':
[0075] oxide of PTH peptide in which residues corresponding to the
position 8 and position 18 methionine of human PTH (1-34) have been
changed into methionine sulfoxide residues;
[0076] 4) analog 4':
[0077] oxide of PTH peptide in which the residue corresponding to
the position 8 methionine of human PTH (1-34) has been changed into
a methionine sulfoxide residue and the residue corresponding to the
position 23 tryptophan has been changed into a residue shown by the
above structural formula (a);
[0078] 5) analog 5':
[0079] oxide of PTH peptide in which the residue corresponding to
the position 18 methionine of human PTH (1-34) has been changed
into a methionine sulfoxide residue and the residue corresponding
to the position 23 tryptophan has been changed into a residue shown
by the above structural formula (a);
[0080] 6) analog 6':
[0081] oxide of PTH peptide in which the residue corresponding to
the position 18 methionine of human PTH (1-34) has been changed
into a methionine sulfoxide residue and the residue corresponding
to the position 23 tryptophan has been changed into a residue shown
by the above structural formula (b);
[0082] 7) analog 7':
[0083] oxide of PTH peptide in which the residue corresponding to
the position 8 methionine of human PTH (1-34) has been changed into
a methionine sulfoxide residue;
[0084] 8) analog 8':
[0085] oxide of PTH peptide in which the residue corresponding to
the position 18 methionine of human PTH (1-34) has been changed
into a methionine sulfoxide residue;
[0086] 9) analog 9':
[0087] oxide of PTH peptide in which the residue corresponding to
the position 23 tryptophan of human PTH (1-34) has been changed
into a residue shown by the above structural formula (a);
[0088] 10) analog 10':
[0089] oxide of PTH peptide in which the residue corresponding to
the position 23 tryptophan of human PTH (1-34) has been changed
into a tryptophan monoxide residue shown by the following
structural formula (c-1) or (c-2);
##STR00003##
or
[0090] 11) analog 11':
[0091] oxide of PTH peptide in which the residue corresponding to
the position 23 tryptophan of human PTH (1-34) has been changed
into a residue shown by the above structural formula (b).
[0092] [4] The freeze-dried preparation containing PTH peptide set
forth in [2] wherein high-purity means that the amount of at least
one of the above analogs 1 to 11 versus the sum of the amount of
PTH peptide and the total amount of PTH analogs in the preparation
is 1.0% or less and/or that the total amount of the above analogs 1
to 11 versus the sum of the amount of PTH peptide and the total
amount of PTH analogs is 5.0% or less.
[0093] [5] The freeze-dried preparation containing PTH peptide set
forth in [3] wherein high-purity means that the amount of at least
one of the above analogs 1' to 11' versus the sum of the amount of
PTH peptide and the total amount of PTH analogs in the preparation
is 1.0% or less and/or that the total amount of the above analogs
1' to 11' versus the sum of the amount of PTH peptide and the total
amount of PTH analogs is 5.0% or less.
[0094] [6] The freeze-dried preparation containing PTH peptide
according to any of [1] to [5] wherein the PTH peptide is human PTH
(1-34).
[0095] [7] The freeze-dried preparation containing PTH peptide
according to any of [1] to [6] wherein the freeze-dried preparation
containing PTH peptide is housed in a glass vial.
[0096] [8] The freeze-dried preparation containing PTH peptide
according to any of [1] to [7], characterized in that exposure of
the solution containing PTH peptide to air environments within a
pharmaceutical production facility prior to freeze drying is
controlled in any one or more steps selected from a step for
preparing a solution containing PTH peptide, an aseptic filtration
step, a drug solution dispensing step, and a step for loading into
a freeze drying means.
[0097] [9] The freeze-dried preparation containing PTH peptide set
forth in [8] characterized in being produced using a method that
also includes control of exposure of the freeze-dried product to
air environments within a pharmaceutical production facility in a
vial sealing step after freeze drying.
[0098] [10] The freeze-dried preparation containing PTH peptide
according to any of [1] to [9] characterized in that exposure of
the solution containing PTH peptide to air environments within a
pharmaceutical production facility prior to freeze drying is
controlled in the step for loading into the freeze drying
means.
[0099] [11] The freeze-dried preparation containing PTH peptide set
forth in [10] characterized in that the above exposure is
controlled by using a freeze-drying chamber equipped with a means
for controlling the entrance of air within a pharmaceutical
production facility into the freeze-drying means.
[0100] [12] The freeze-dried preparation containing PTH peptide set
forth in [11] characterized in that the freeze-drying means is a
freeze-drying chamber having an easily openable and closable
sub-door provided in an opening created in a small door unit opened
when containers housing the solution containing PTH peptide prior
to freeze drying are loaded into and unloaded from this means,
thereby controlling exposure of the solution containing PTH peptide
to air environments within a pharmaceutical production facility
prior to freeze drying by opening this sub-door only during
container loading and quickly closing the sub-door after
loading.
[0101] [13] A freeze-dried preparation containing PTH peptide set
forth in [11] wherein the freeze-drying means is a freeze-drying
chamber having an opening created in a small door unit opened when
containers housing the solution containing PTH peptide prior to
freeze drying are loaded into and unloaded from this means, and the
means for controlling the ingress of air within a pharmaceutical
production facility into the freeze-drying means is an
airflow-adjusting cover that can change the air flow to a direction
not directed from this opening to the inside of the chamber.
[0102] [14] The freeze-dried preparation containing PTH peptide set
forth in [10] characterized in that the loading step controls
exposure of the solution containing PTH peptide to air environments
within a pharmaceutical production facility prior to freeze drying
by purging the inside of the freeze-drying means with an inert
gas.
[0103] [15] The freeze-dried preparation containing PTH peptide set
forth in [10] wherein the freeze-drying means is a freeze-drying
chamber having an easily openable and closable sub-door provided in
an opening created in a small door unit opened when containers
housing the solution containing PTH peptide prior to freeze drying
are loaded into and unloaded from this means, thereby controlling
exposure of the solution containing PTH peptide to air environments
within a pharmaceutical production facility prior to freeze drying
by opening this sub-door only during container loading and quickly
closing the sub-door after loading and purging the inside of the
freeze-drying means with an inert gas in the loading step.
[0104] [16] The freeze-dried preparation containing PTH peptide set
forth in [10] wherein the freeze-drying means is a freeze-drying
chamber having an opening created in a small door unit opened when
containers housing the solution containing PTH peptide prior to
freeze drying are loaded into and unloaded from this means, this
opening being equipped with an airflow-adjusting cover, thereby
controlling exposure of the solution containing PTH peptide to air
environments within a pharmaceutical production facility prior to
freeze drying by changing the direction of the airflow-adjusting
cover so that air is not directed into the chamber and purging the
inside of the freeze-drying means with an inert gas in the loading
step.
[0105] [17] A freeze-dried preparation containing PTH peptide
according to any of [10] to [16] wherein the loading step is a step
that spans three or more hours.
[0106] [18] The freeze-dried preparation containing PTH peptide
according to any of [8] to [17] wherein time from the beginning of
the step for preparing a solution containing PTH peptide to the end
of the step for loading into the freeze-drying means spans three or
more hours, and production is performed using a method for
controlling exposure of the solution containing PTH peptide to an
air environment within a pharmaceutical production facility in one
or more steps during the time.
[0107] [19] A freeze-dried preparation containing PTH peptide
according to any of [14] to [18] wherein the inert gas is nitrogen
gas.
[0108] [20] A freeze-dried preparation containing high-purity PTH
peptide as an active ingredient, the PTH-peptide-containing
freeze-dried preparation manufactured using a method characterized
in that exposure of a solution containing PTH peptide to air
environments within a pharmaceutical production facility prior to
freeze drying is controlled during loading into the freeze-drying
means; wherein "high-purity" means at least that the amount of at
least one PTH analog versus the sum of the amount of PTH peptide
and the total amount of PTH analogs in the preparation is 1.0% or
less and/or that the total amount of PTH analogs versus the sum of
the amount of PTH peptide and the total amount of PTH analogs is
5.0% or less; the loading step is a step that spans three or more
hours; the air environment is an environment that maintains one-way
air flow of clean air that has passed through an HEPA filter
downward from above; and the velocity of the air flow 20 cm
directly under the HEPA filter is 0.2-1.0 m/s.
[0109] [21] A method for producing a freeze-dried preparation
containing PTH peptide, the method being characterized in that
exposure of the solution containing PTH peptide to air environments
within a pharmaceutical production facility is controlled in one or
more steps from the beginning of the step for preparing a solution
containing PTH peptide to the end of the step for loading into a
freeze-drying means.
[0110] [22] The method set forth in [21] wherein exposure of the
freeze-dried product to air environments within a pharmaceutical
production facility is also controlled in the step for sealing
vials after freeze drying.
[0111] [23] The method set forth in [21] or [22] characterized in
that exposure of the solution containing PTH peptide to air
environments within a pharmaceutical production facility is
controlled in the step for loading into a freeze-drying means.
[0112] [24] The method set forth in [23] characterized in that
exposure is controlled using a freeze-drying chamber equipped with
means for controlling the inflow of air within a pharmaceutical
production facility into the freeze-drying means.
[0113] [25] The method set forth in [23] or [24] wherein the step
for loading into the freeze-drying means is a step that spans three
or more hours.
[0114] [26] The method set forth in [24] or [25] wherein the
freeze-drying means is a freeze-drying chamber having an easily
openable and closable sub-door provided in an opening created in a
small door unit opened when containers housing the solution
containing PTH peptide prior to freeze drying are loaded and
unloaded, thereby controlling exposure of the solution containing
PTH peptide to air environments within a pharmaceutical production
facility prior to freeze drying by opening this sub-door only
during container loading and quickly closing the sub-door after
loading.
[0115] [27] The method set forth in [24] or [25] wherein the
freeze-drying means is a freeze-drying chamber having an opening
created in a small door unit opened when containers housing the
solution containing PTH peptide prior to freeze drying are loaded
into and unloaded from this means, and the means for controlling
the ingress of air within a pharmaceutical production facility into
the freeze-drying means is an airflow-adjusting cover that can
change the air flow to a direction not directed from this opening
to the inside of the chamber.
[0116] [28] The method set forth in [23] or [25] characterized in
that exposure of the solution containing PTH peptide to air
environments within a pharmaceutical production facility prior to
freeze drying is controlled by purging the inside of the
freeze-drying means with an inert gas.
[0117] [29] The method set forth in [23] or [25] characterized in
that the freeze-drying means is a freeze-drying chamber having an
easily openable and closable sub-door provided in an opening
created in a small door unit opened when containers housing the
solution containing PTH peptide prior to freeze drying are loaded
and unloaded, thereby controlling exposure of the solution
containing PTH peptide to air environments within a pharmaceutical
production facility prior to freeze drying by opening this sub-door
only during container loading and quickly closing the sub-door
after loading and purging the inside of the freeze-drying means
with an inert gas in the loading step.
[0118] [30] The method set forth in [23] or [25] characterized in
that the freeze-drying means is a freeze-drying chamber having an
opening created in a small door unit opened when containers housing
the solution containing PTH peptide prior to freeze drying are
loaded into and unloaded from this means, this opening being
equipped with an airflow-adjusting cover, thereby controlling
exposure of the solution containing PTH peptide to air environments
within a pharmaceutical production facility prior to freeze drying
by changing the airflow-adjusting cover to a direction in which the
air flow is not directed into the chamber and purging the inside of
the freeze-drying means with an inert gas in the loading step.
[0119] [31] The method set forth in any of [28] to [30] wherein the
inert gas is nitrogen.
[0120] [32] The method set forth in any of [21] to [31] wherein the
container housing the solution containing PTH peptide is a glass
vial.
[0121] [33] The method set forth in any of [21] to [32] wherein the
PTH is human PTH (1-34).
[0122] [34] The method set forth in any of [21] to [33] wherein the
air environment within a pharmaceutical production facility is an
air environment in which 1) the air is of grade A, 2) clean air
that has passed through an HEPA filter having the ability to trap
particles having a particle size of 0.3 .mu.m at an efficiency of
99.97% or higher is maintained as a one-way air flow downward from
above, and 3) the ozone concentration is 0.001-0.1 ppm.
[0123] [35] The method set forth in any of [21] to [34] wherein the
air environment within a pharmaceutical production facility is an
air environment containing a formaldehyde concentration of 0.1 ppm
or less.
[0124] [36] The method set forth in any of [21] to [35] wherein the
amount of at least one PTH analog versus the sum of the amount of
PTH peptide and total amount of PTH analogs is 1.0% or less and/or
the total amount of PTH analogs versus the sum of the amount of PTH
peptide and total amount of PTH analogs is 5.0% or less in the
freeze-dried preparation containing PTH peptide.
[0125] [37] The method set forth in any of [21] to [36] to control
the production of PTH analogs 1 to 11 set forth in [2].
[0126] [38] The method set forth in any of [21] to [36] to control
the production of PTH analogs 1' to 11' set forth in [3].
[0127] [39] A freeze-dried preparation containing PTH peptide
manufactured using the method according to any of [21]-[38].
[0128] [40] A method for producing a freeze-dried preparation
containing high-purity PTH peptide as an active ingredient, the
method characterized in that exposure of a solution containing PTH
peptide to air environments within a pharmaceutical production
facility prior to freeze drying is controlled during loading into
the freeze-drying means; wherein "high-purity" means at least that
the amount of at least one PTH analog versus the sum of the amount
of PTH peptide and the total amount of PTH analogs is 1.0% or less
and/or that the total amount of PTH analogs versus the sum of the
amount of PTH peptide and the total amount of PTH analogs is 5.0%
or less in the preparation; the loading step is a step that spans
three or more hours; the air environment is an environment in which
clean air that has passed through an HEPA filter is maintained as a
one-way air flow downward from above; and the velocity of the air
flow 20 cm directly under the HEPA filter is 0.2-1.0 m/s.
[0129] The present invention also intends a test method that is
important for compliance with laws and regulations and assurance of
compatibility of the freeze-dried preparation containing PTH
peptide as a pharmaceutical. This test method is characterized in
confirming the presence of any one or more or all of the above PTH
analogs and/or determines the amounts present. The following are
also encompassed as aspects and preferred embodiments.
[0130] [41] A method for testing a freeze-dried preparation
containing PTH peptide, the method being characterized in
confirming the presence of at least one or more of the PTH analogs
1 to 11 of [2] and/or determining the amounts present in the
freeze-dried preparation containing PTH peptide.
[0131] [42] A method for testing a freeze-dried preparation
containing PTH peptide, the method being characterized in
confirming the presence of at least one or more of the PTH analogs
1' to 11' of [3] and/or determining the amounts present in the
freeze-dried preparation containing PTH peptide.
[0132] [43] The method set forth in [41] or [42] wherein
determination of the PTH analogs includes calculating the area of
the peak corresponding to the PTH analog on a chromatogram when the
ultraviolet absorption of a sample derived from a freeze-dried
preparation containing PTH peptide is measured by high-performance
liquid chromatography.
[0133] [44] The method set forth in [43] including calculation of
the purity of the PTH peptide in the freeze-dried preparation
containing PTH peptide by comparing the area of a peak
corresponding to a PTH analog on a chromatogram and the peak area
corresponding to PTH peptide or the sum of the peak area of PTH
peptide and the peak area of all other PTH analogs detected on the
same chromatogram when the ultraviolet absorbance of a sample
derived from a freeze-dried preparation containing PTH peptide is
measured by high-performance liquid chromatography.
[0134] [45] The method set forth in [44] including calculation of
the purity of the PTH peptide in a freeze-dried preparation
containing PTH peptide by comparing the area of that peak and the
peak area corresponding to PTH peptide or the sum of the peak area
of PTH peptide and the peak area of all other PTH analogs detected
on the same chromatogram when using chromatography conditions such
that any two or more PTH analogs are detected as one or more single
peaks on the chromatogram.
[0135] [46] The method set forth in any of [41] to [45] for
ensuring that the amount of at least one PTH analog versus the sum
of the amount of PTH peptide and the total amount of PTH analogs is
1.0% or less and/or the total amount of PTH analogs versus the sum
of the amount of PTH peptide and the total amount of PTH analogs is
5.0% or less in a freeze-dried preparation containing PTH
peptide.
[0136] [47] The method set forth in any of [41] to [46] including
the detection of the mass number of the PTH analogs using a
high-performance liquid chromatograph-mass spectrometer.
[0137] [48] The method set forth in any of [41] to [47] including
fractionating a substance that gives a single peak on the
chromatogram and identifying the mass number of the fragments
produced by digesting this substance using trypsin.
[0138] [49] A method for producing a pharmaceutical comprising a
freeze-dried preparation containing PTH peptide including a step to
carry out the test method of any of [41] to [48].
[0139] The following aspects are also intended as preferred
freeze-dried preparations containing PTH peptide of the present
invention.
[0140] [50] A freeze-dried preparation containing PTH peptide, the
freeze-dried preparation containing PTH peptide being characterized
in that at least one or more PTH analogs is 1.0% or less versus the
sum of the amount of PTH peptide and the total amount of PTH
analogs and/or the total amount of PTH analogs is 5.0% or less
versus the sum of the amount of PTH peptide and the total amount of
PTH analogs.
[0141] [51] A freeze-dried preparation containing PTH peptide, the
freeze-dried preparation containing PTH peptide being characterized
in that any of the respective PTH analogs is 1.0% or less versus
the sum of the amount of PTH peptide and the total amount of PTH
analogs and/or the total amount of PTH analogs is 5.0% or less
versus the sum of the amount of PTH peptide and the total amount of
PTH analogs.
[0142] [52] A freeze-dried preparation containing PTH peptide set
forth in [50] or [51] wherein the PTH analog is an analog set forth
in [2].
[0143] [53] A freeze-dried preparation containing PTH peptide set
forth in [50] or [51] wherein the PTH analog is an analog set forth
in [3].
[0144] [54] A freeze-dried preparation containing PTH peptide set
forth in [52] wherein the amount of analogs versus the sum of the
amount of PTH peptide and the total amount of PTH analogs is in at
least the following relationships:
[0145] the amount of analog 1 is 0.04% or less;
[0146] the total amount of analog 3 and analog 4 is 0.11% or
less;
[0147] the amount of analog 5 is 0.26% or less;
[0148] the amount of analog 7 is 0.33% or less;
[0149] the amount of analog 8 is a percentage selected arbitrarily
from 0.21-1.00%; and
[0150] the amount of analog 9 is 0.68% or less.
[0151] [55] A freeze-dried preparation containing PTH peptide set
forth in [53] wherein the amount of analogs versus the sum of the
amount of PTH peptide and the total amount of PTH analogs is in at
least the following relationships:
[0152] the amount of analog 1' is 0.04% or less;
[0153] the total amount of analog 3' and analog 4' is 0.11% or
less;
[0154] the amount of analog 5' is 0.26% or less;
[0155] the amount of analog 7' is 0.33% or less;
[0156] the amount of analog 8' is a percentage selected arbitrarily
from 0.21-1.00%; and
[0157] the amount of analog 9' is 0.68% or less.
[0158] [56] A freeze-dried preparation containing PTH peptide set
forth in [52] wherein the amount of analog 1, amount of analog 2,
total amount of analog 3 and analog 4, amount of analog 5, amount
of analog 6, amount of analog 7, amount of analog 8, amount of
analog 9, and total amount of analog 10 and analog 11 are all 1.0%
or less versus the sum of the amount of PTH peptide and the total
amount of PTH analogs.
[0159] [57] A freeze-dried preparation containing PTH peptide set
forth in [53] wherein the amount of analog 1', amount of analog 2',
total amount of analog 3' and analog 4', amount of analog 5',
amount of analog 6', amount of analog 7', amount of analog 8',
amount of analog 9', and total amount of analog 10' and analog 11'
are all 1.0% or less versus the sum of the amount of PTH peptide
and the total amount of PTH analogs.
[0160] [58] A freeze-dried preparation containing PTH peptide
according to any of [50] to [57] wherein the freeze-dried
preparation containing PTH peptide is a preparation housed in a
stoppered glass container.
[0161] [59] A freeze-dried preparation containing PTH peptide
according to any of [50] to [58] wherein the freeze-dried
preparation containing PTH peptide is a glass vial preparation.
[0162] [60] A freeze-dried preparation containing PTH peptide
according to any of [50] to [59] wherein the PTH peptide is human
PTH (1-34)
[0163] A freeze-dried preparation containing high-purity PTH is
provided by the present invention. Specifically, the production of
PTH analogs characterized and confirmed to be produced in the
freeze-dried preparation containing PTH which is undesirable during
pharmaceutical manufacture is inhibited and decreased in the
present invention. A preparation qualified as a pharmaceutical can
also be produced while confirming and assuring the quality of the
freeze-dried preparation containing PTH simply, rapidly, and
accurately by determining the PTH analogs.
BRIEF DESCRIPTION OF THE DRAWINGS
[0164] FIG. 1 shows the chromatograms when the ultraviolet (214 nm)
absorbance was measured by high-performance liquid chromatography
(HPLC) taking as the sample a PTH peptide used as a raw material of
a PTH peptide freeze-dried preparation produced as a working
example and comparative example. The horizontal axis represents the
time (min), and the vertical axis represents the absorption
intensity. The large peak appearing at approximately 20-21 minutes
is human PTH (1-34). "6 (encircled number)" corresponds to analog 7
(analog 7'), and "7 (encircled number)" corresponds to analog 8
(analog 8').
[0165] FIG. 2 shows the chromatogram when the ultraviolet (214 nm)
absorbance was measured by high-performance liquid chromatography
(HPLC) taking as the sample a PTH peptide freeze-dried preparation
produced as Example 1. The horizontal axis represents the time
(min), and the vertical axis represents the absorption intensity.
The large peak appearing at 21.157 min (retention time) is human
PTH (1-34). "1 (encircled number)" corresponds to analog 1 (analog
1'); "2 (encircled number)" corresponds to analog 2 (analog 2'); "3
(encircled number)" corresponds to a mixture of analog 3 and analog
4' (mixture of analog 3' and analog 4'); "4 (encircled number)"
corresponds to analog 5 (analog 5'); "6 (encircled number)"
corresponds to analog 7 (analog 7'); "7 (encircled number)"
corresponds to analog 8 (analog 8'); "8 (encircled number)"
corresponds to analog 9 (analog 9'); "9 (encircled number)"
corresponds to a mixture of analog 10 and analog 11 (mixture of
analog 10' and mixture 11').
[0166] FIG. 3 shows the chromatogram when the ultraviolet (214 nm)
absorbance was measured by high-performance liquid chromatography
(HPLC) taking as the sample a PTH peptide freeze-dried preparation
produced as Comparative Example 1. The horizontal axis represents
the time (min), and the vertical axis represents the absorption
intensity. The large peak appearing at 20.279 min (retention time)
is human PTH (1-34). "5 (encircled number)" corresponds to analog 6
(analog 6'); the meaning of the other encircled numbers is the same
as in FIG. 2.
[0167] FIG. 4 shows the chromatogram when the ultraviolet (214 nm)
absorbance was measured by high-performance liquid chromatography
(HPLC) taking as the sample a PTH peptide freeze-dried preparation
exposed to ozone as in Test Example 2. The horizontal axis
represents the time (min), and the vertical axis represents the
absorption intensity. The large peak appearing at 22.670 min
(retention time) is human PTH (1-34). The meaning of the encircled
numbers is the same as in FIG. 2.
[0168] FIG. 5 shows the structure of a methionine oxide
compound.
[0169] FIG. 6 shows the structure of a tryptophan variant.
[0170] FIG. 7 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 1. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0171] FIG. 8 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 2. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0172] FIG. 9 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of a mixture of analog 3
and analog 4. The horizontal axis represents the time (min), and
the vertical axis represents the detection intensity.
[0173] FIG. 10 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 5. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0174] FIG. 11 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 6. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0175] FIG. 12 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 7. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0176] FIG. 13 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 8. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0177] FIG. 14 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of analog 9. The
horizontal axis represents the time (min), and the vertical axis
represents the detection intensity.
[0178] FIG. 15 shows the results of high-performance liquid
chromatography-mass spectrometry (LC/MS) of a mixture of analogs 10
and 11. The horizontal axis represents the time (min), and the
vertical axis represents the detection intensity.
[0179] FIG. 16 is a schematic diagram showing an example of
preferred freeze-drying means of the present invention.
[0180] FIG. 17 is a schematic diagram showing an example of
preferred freeze-drying means of the present invention.
[0181] The present invention shall now be described in further
detail.
[0182] (1) PTH Peptide
[0183] The term "PTH peptide" in the present invention is used as a
collective term for natural PTH and substances of equivalent
physiologic activity. The physiologic activity of PTH is
characterized as acting to raise the serum calcium. Preferred PTH
peptides encompass natural PTH and partial peptides thereof, which
may be peptides having a molecular weight of from about 4000 to
10,000. However, PTH peptides are those wherein any of the
constituent amino acid residues have not been chemically modified
at all in comparison to the natural form; they do not include the
(2) PTH analogs discussed later. Concrete examples of partial
peptides include human PTH (1-34), human PTH (1-35), human PTH
(1-36), human PTH (1-37), human PTH (1-38), human PTH (1-84), and
the like, all of which have a sequence of 34-84 amino acids.
Specifically, human PTH (1-34) is a partial peptide of the natural
form sequence corresponding to amino acids numbers 1-34 of natural
human PTH. Human PTH (1-34) and human PTH (1-84) are preferred, and
human PTH (1-34) is especially preferred. The amino acid sequence
of human PTH (1-34) is as follows:
TABLE-US-00001 [Chemical Formula 4]
H-Ser-Val-Ser-Glu-Ile-Gln-Leu-Met- (SEQ ID NO: 4)
His-Asn-Leu-Gly-Lys-His-Leu-Asn- Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-
Arg-Lys-Lys-Leu-Gln-Asp-Val-His- Asn-Phe-OH
[0184] The PTH peptide of the present invention may also be present
as a salt formed with one or more volatile organic acids. Examples
of volatile organic acids include trifluoroacetic acid, formic
acid, acetic acid, and the like. Acetic acid can be given as a
preferred example, but preferred examples are not limited to this.
The ratio of the two when free PTH peptide and a volatile organic
acid form a salt is not particularly restricted as long as a salt
forms. For example, since human PTH (1-34) has nine basic amino
acid residues and four acidic amino acid residues in its molecule,
taking into consideration salt formation in these molecules, the
basic amino acid 5 residue can be made into a chemical equivalent
of acetic acid. For example, if an acetic acid content represented
by the acetic acid weight.times.100(%)/peptide weight of human PTH
(1-34) is used as the amount of acetic acid, as one theory, the
chemical equivalent of acetic acid versus free human PTH (1-34)
becomes approximately 7.3% (wt %). In this specification, free
human PTH (1-34) sometimes is also called "teriparatide," and the
acetate of teriparatide is sometimes also called "teriparatide
acetate." The acetic acid content in teriparatide acetate is not
particularly restricted as long the teriparatide and acetic acid
form a salt. For example, it may be 7.3%, which is the above
theoretical chemical equivalent, or higher, or it may be from more
than 0% to less than 1%. More concrete examples of the acetic acid
content in teriparatide acetate are 1-7%, preferably 2-6%.
[0185] However, regardless of whether the PTH peptide of the
present invention is a free compound or a salt thereof, the amount
of PTH peptide in the preparation of the present invention, amount
of various PTH analogs, amount of PTH analog mixture, and total PTH
analogs can be determined by HPLC testing. It should be noted that
in this case the PTH peptide and PTH analogs are all determined as
free compounds.
[0186] (2) PTH Analogs
[0187] The term "PTH analog" in the present invention is defined in
the broad sense as one detected as a peak different from the PTH
peptide which is the active ingredient on the chromatogram when a
sample from a freeze-dried preparation containing PTH peptide is
subjected to HPLC. Therefore, if detected as one peak different
from the original PTH peptide on the chromatogram, all of the
chemical substances included in this peak may be regarded together
as a single "PTH analog" even when two or more separate chemical
substances are present in mixture within the peak. That is, for the
purposes of general measurement and confirming the purity of a
freeze-dried preparation, even a mixture of multiple chemical
substances detectable as a single peak on the HPLC chromatogram is
comprehensively termed an "analog" and confirmation of purity,
calculation of purity, and the like are broadly performed regarding
a single peak consisting of such a mixture as one "analog" for the
sake of convenience. There is consequently no problem with
regarding a mixture of multiple chemical substances detected as a
single peak in HPLC under given conditions comprehensively as one
type of "PTH analog."
[0188] In the present invention, the PTH analogs discovered to be
produced during the production of a freeze-dried preparation
containing PTH peptide were characterized as shown in Table 1
below.
TABLE-US-00002 TABLE 1 Characterization of PTH analogs Fragment
Overview of changed Amino changes (trypsin acid Change Nature of
Estimated structural Analog digestion) changed in mass change
changes 1 T1 Met 8 16 Da Oxidation Human PTH (1-34)-Met 8 T2 Met 18
16 Da Oxidation [O]-Met 18 [O]-Trp 23 T3 Trp 23 32 Da a)
[dioxidation] 2 T1 Met 8 16 Da Oxidation Human PTH (1-34)-Met 8 T2
Met 18 16 Da Oxidation [O]-Met 18 [O]-Trp 23 T3 Trp 23 4 Da b)
[dioxidation-formic acid elimination] 3 T1 Met 8 16 Da Oxidation
Human PTH (1-34)-Met 8 T2 Met 18 16 Da Oxidation [O]-Met 18 [O] 4
T1 Met 8 16 Da Oxidation Human PTH (1-34)-Met 8 T3 Trp 23 32 Da a)
[O]-Trp 23 [dioxidation] 5 T2 Met 18 16 Da Oxidation Human PTH
(1-34)-Met 18 T3 Trp 23 32 Da a) [O]-Trp 23 [dioxidation] 6 T2 Met
18 16 Da Oxidation Human PTH (1-34)-Met 18 T3 Trp 23 4 Da b)
[O]-Trp 23 [dioxidation-formic acid elimination] 7 T1 Met 8 16 Da
Oxidation Human PTH (1-34)-Met 8 [O] 8 T2 Met 18 16 Da Oxidation
Human PTH (1-34)-Met 18 [O] 9 T3 Trp 23 32 Da a) Human PTH
(1-34)-Trp 23 [dioxidation] 10 T3 Trp 23 16 Da c) Human PTH
(1-34)-Trp 23 [monoxidation] 11 T3 Trp 23 4 Da b) Human PTH
(1-34)-Trp 23 [dioxidation-formic acid elimination]
[0189] In the above Table 1, T1-T3 are typical fragments produced
when each analog is digested by trypsin and are as follows when
listed based on the amino acid sequence of the human PTH (1-34)
sequence.
TABLE-US-00003 [Chemical Formula 5] T1: (corresponding to positions
1-13 of human PTH (1-34)
Ser-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu- Gly-Lys (SEQ ID NO: 1)
T2: (corresponding to positions 14-20 of human PTH (1-34)
His-Leu-Asn-Ser-Met-Glu-Arg (SEQ ID NO: 2) T3: (corresponding to
positions 21-25 of human PTH (1-34) Val-Glu-Trp-Leu-Arg (SEQ ID NO:
3)
[0190] The numbers of the amino acids changed in Table 1 are
expressed as the corresponding amino acid numbers of the human PTH
(1-34) sequence. The same notation is used in this specification
unless otherwise indicated.
[0191] In the estimated structures of Table 1, human PTH (1-34)-Met
8 [O]-Met 18 [O]-Trp 23 [dioxidation] (analog 1') means a PTH
analog in which the residues corresponding to the position 8 and 18
methionine of human PTH (1-34) are each methionine sulfoxide
residues, the residue corresponding to the position 23 tryptophan
is a residue shown by the following structure (a) (Trp 23 oxidation
(a) residue), and the other structures are the same as the original
PTH peptide.
##STR00004##
[0192] Human PTH (1-34)-Met 8 [O]-Met 18 [O]-Trp 23
[dioxidation-formic acid elimination] (analog 2') means a PTH
analog in which the residues corresponding to the position 8 and 18
methionine of human PTH (1-34) are each methionine sulfoxide
residues, the residue corresponding to the position 23 tryptophan
is a residue shown by the following structure (b) (Trp 23 oxidation
(b) residue), and the other structures are the same as the original
PTH peptide.
##STR00005##
[0193] Similarly, human PTH (1-34)-Met 8 [O]-Met 18 [O] (analog 3')
means a PTH analog in which residues corresponding to the position
8 and 18 methionine of human PTH (1-34) are each methionine
sulfoxide residues and the other structures are the same as the
original PTH peptide. Human PTH (1-34)-Met 8 [O]-Trp 23
[dioxidation] (analog 4') means a PTH analog in which the residue
corresponding to the position 8 methionine of human PTH (1-34) is a
methionine sulfoxide residue, the residue corresponding to the
position 23 tryptophan is a Trp 23 oxidation (a) residue, and the
other structures are the same as the original PTH peptide.
Furthermore, analog 3' and analog 4' tend to be detected as a
single peak, depending on the HPLC conditions. In this case, the
PTH analog may be defined as a mixture of analog 3' and analog 4'
as discussed above.
[0194] Human PTH (1-34)-Met 18 [O]-Trp 23 [dioxidation] (analog 5')
means a PTH analog in which the residue corresponding to the
position 18 methionine of human PTH (1-34) is a methionine
sulfoxide residue, the residue corresponding to the position 23
tryptophan is a Trp 23 oxidation (a) residue, and the other
structures are the same as the original PTH peptide.
[0195] Human PTH (1-34)-Met 18 [O]-Trp 23 [dioxidation-formic acid
elimination] (analog 6') means a PTH analog in which the residue
corresponding to the position 18 methionine of human PTH (1-34) is
a methionine sulfoxide residue, the residue corresponding to the
position 23 tryptophan is a Trp 23 oxidation (b) residue, and the
other structures are the same as the original PTH peptide.
[0196] Human PTH (1-34)-Met 8 [O] (analog 7') means a PTH analog in
which the residue corresponding to the position 8 methionine of
human PTH (1-34) is a methionine sulfoxide residue and the other
structures are the same as the original PTH peptide.
[0197] Human PTH (1-34)-Met 18 [O] (analog 8') means a PTH analog
in which the residue corresponding to the position 18 methionine of
human PTH (1-34) is a methionine sulfoxide residue and the other
structures are the same as the original PTH peptide.
[0198] Human PTH (1-34)-Trp 23 [dioxidation] (analog 9') means a
PTH analog in which the residue corresponding to the position 23
tryptophan of human PTH (1-34) is a Trp 23 oxidation (a) residue
and the other structures are the same as the original PTH
peptide.
[0199] Human PTH (1-34)-Trp 23 [monoxidation] (analog 10') means a
PTH analog in which the residue corresponding to the position 23
tryptophan of human PTH (1-34) is a residue shown by the following
structure (c)-1 or (c)-2 (Trp 23 oxidation (c) residue) residue and
the other structures are the same as the original PTH peptide.
##STR00006##
[0200] Human PTH (1-34)-Trp 23 [dioxidation-formic acid
elimination] (analog 11') means a PTH analog in which the residue
corresponding to the position 23 tryptophan of human PTH (1-34) is
a Trp 23 oxidation (b) residue and the other structures are the
same as the original PTH peptide. Furthermore, analog 10' and
analog 11' tend to be detected as a single peak, depending on the
HPLC conditions. In this case, the PTH analog may be defined as a
mixture of analog 10' and analog 11' as discussed above
[0201] In the above analogs 1' to 11', the PTH peptide changes in
that modified amino acid residues produced by oxidation of either
methionine or tryptophan are introduced. It is therefore logical to
assume that the production of the PTH analogs of the present
invention is begun by contact between a substance having oxidizing
capability and the PTH peptide. In this specification, a "substance
having oxidizing capability" means a substance having the capacity
to oxidize a structural amino acid of the PTH peptide, especially
methionine or tryptophan. Given that ozone, formaldehyde, and other
such oxidizing gaseous molecules are sometimes present in the air
inside a pharmaceutical production facility, as discussed above,
such substances capable of oxidizing methionine and tryptophan
which may be contained in the air inside a pharmaceutical
production facility are of interest as "substances having oxidizing
capability" in this specification.
[0202] Furthermore, as is evident from the above, the above
definition of PTH analogs can be applied even when the PTH peptide
contained as an active ingredient is other than human PTH (1-34).
For example, when human PTH (1-84) is used as an active ingredient,
the corresponding analog 1' can also be expressed as human PTH
(1-84)-Met 8 [O]-Met 18 [O]-Trp 23 [dioxidation]. In this case, the
analog can be specified as one in which the position 8 and 18
methionine residues of human PTH (1-84) are each methionine
sulfoxide residues, the position 23 tryptophan residue is a Trp 23
oxidation (a) residue, and the other structures are the same as
human PTH (1-84).
[0203] (3) Detection and Determination of PTH Analogs
[0204] The PTH analogs in a freeze-dried preparation containing PTH
can be detected or determined by producing a sample by dissolving
the preparation in a suitable solvent (phosphate buffer containing
benzalkonium chloride or the like) and subjecting this sample to
HPLC under, for example, the following conditions.
[0205] <HPLC Conditions>
[0206] a) Detector: Ultraviolet absorptiometer (measurement
wavelength: 214 nm)
[0207] b) Column: Stainless steel tube 150 mm long having an inner
diameter of 4.6 mm packed with 3.5 .mu.m of octadecylsilylated
silica gel for liquid chromatography (Zorbax 300SB-C18 manufactured
by Agilent Technologies or an equivalent product)
[0208] c) Column temperature: Constant temperature near 40.degree.
C.
[0209] d) Mobile phase:
[0210] Mobile phase A: Dissolve 28.4 g of anhydrous sodium sulfate
in 900 mL of water and bring to 1000 mL by adding water after
adding phosphoric acid to adjust the pH to 2.3. Add 100 mL of
acetonitrile to this 900 mL of liquid.
[0211] Mobile phase B: Dissolve 28.4 g of anhydrous sodium sulfate
in 900 mL of water and bring to 1000 mL by adding water after
adding phosphoric acid to adjust the pH to 2.3 Add 500 mL of
acetonitrile to 500 mL of this liquid.
[0212] e) Mobile phase feed: Concentration gradient control is
provided by varying the mixture ratio of mobile phase A and mobile
phase B as shown in Table 2.
TABLE-US-00004 TABLE 2 Concentration gradient control Time after
sample injection (min) Mobile phase A (vol %) Mobile phase B (vol
%) 0-5 100.fwdarw.65 0.fwdarw.35 5-35 65.fwdarw.60 35.fwdarw.40
35-45 60.fwdarw.0 40.fwdarw.100
[0213] f) Flow rate: 1.0 mL/min
[0214] g) Detection time: 45 minutes after injection of the sample
solution. However, this is from the back of the solvent peak.
[0215] Since the PTH peptide and PTH analogs of the present
invention have substantial absorbance in the ultraviolet region,
monitoring the ultraviolet absorption is advantageous for their
detection and determination. The measurement wavelength is not
particularly restricted as long as it allows detection of the PTH
peptide and PTH analogs. One or more wavelengths may be selected,
for example, in the range of 210-360 nm, preferably 210-280 nm, and
more preferably 210-254 nm. One example of a suitable wavelength is
214 nm. A chromatogram can be produced based on the measured values
of this ultraviolet absorption.
[0216] The amount of each PTH analog and the amount of PTH peptide
can be determined by calculating each peak area (e.g., by automatic
integration) on the chromatogram based on the chromatogram obtained
by performing HPLC as discussed above. The amount of each PTH
analog (%) and the total amount of PTH analogs (%) can then be
determined and compared based on the calculated values by the
following formulas 1 and 2. Furthermore, the "total peak area" in
the formulas is a value determined by adding the peak area of PTH
peptide and the peak areas of all other PTH analogs detected on the
chromatogram. Therefore, the "total peak area" corresponds to the
"sum of the amount of PTH peptide and total amount of PTH analogs."
In addition, unless specifically indicated otherwise, "%" has the
meaning of the following formula in the present invention.
Amount of each PTH analog (%)=(peak area of each analog/total peak
area).times.100 <Formula 1>
Total amount of PTH analogs (%)=(sum total of peak areas of each
analog/total peak area).times.100 <Formula 2>
[0217] Furthermore, analogs 3 and 4 (analogs 3' and 4') produced
from human PTH (1-34) elute as a single peak, as was mentioned
above, when HPLC is carried out under the above conditions. Since
regarding this single peak as one analog does not affect the
results when used to confirm the purity or measure the preparation
in this case, the mixed peak of analogs 3 and 4 (analogs 3' and 4')
may be regarded as one analog. The same is also true of analogs 10
and 11 (analogs 10' and 11').
[0218] Table 3 below shows a typical measurement example when HPLC
was performed under the above conditions on a sample derived from a
freeze-dried preparation containing human PTH (1-34). Furthermore,
the notation "approximate relative retention time" in the table is
because the relative retention time also sometimes changes
depending on the column used or the mobile phase flow rate.
Nonetheless, each analog can be identified and determined based on
the pattern of the chromatogram taking this relative retention time
as a criterion even in this case.
TABLE-US-00005 TABLE 3 HPLC measurement example Approximate
relative retention time, taking the retention time PTH analog of
human PTH (1-34) as 1.00 (1) Human PTH (1-34)-Met 8 [O]-Met 0.49 18
[O]-Trp 23 [dioxidation] (2) Human PTH (1-34)-Met 8 [O]-Met 0.50 18
[O]-Trp 23 [dioxidation-formic acid elimination] (3) Mixture
containing human PTH (1- 0.52 34)-Met 8 [O]-Met 18 [O] and human
PTH (1-34)-Met 8 [O]- Trp 23 [dioxidation] (4) Human PTH (1-34)-Met
18 [O]- Trp 0.55 23 [dioxidation] (5) Human PTH (1-34)-Met 18 [O]-
Trp 0.57 23 [dioxidation-formic acid elimination] (6) Human PTH
(1-34)-Met 8 [O] 0.60 (7) Human PTH (1-34)-Met 18 [O] 0.66 (8)
Human PTH (1-34)-Trp 23 0.69 [dioxidation] (9) Mixture containing
human PTH (1- 0.74 34)-Trp 23 [monoxidation] and human PTH
(1-34)-Trp 23 [dioxidation- formic acid elimination]
[0219] (4) Freeze-Dried Preparation Containing PTH Peptide
[0220] A freeze-dried preparation containing PTH peptide of the
present invention means a freeze-dried preparation containing PTH
peptide as an active ingredient.
[0221] One embodiment of the freeze-dried preparation containing
PTH peptide of the present invention is a freeze-dried preparation
containing PTH peptide wherein the amount of a PTH analog in the
preparation is 1.0% or less versus the "sum of the amount of PTH
peptide and total amount of PTH analogs" and/or the total amount of
PTH analogs in the preparation is 5.0% or lower versus the "sum of
the amount of PTH peptide and total amount of PTH analogs."
[0222] Another embodiment of the freeze-dried preparation
containing PTH peptide of the present invention is a freeze-dried
preparation containing PTH peptide wherein the amount of any of the
respective PTH analogs is 1.0% or less versus the "sum of the
amount of PTH peptide and total amount of PTH analogs" and/or the
total amount of PTH analogs in the preparation is 5.0% or lower
versus the "sum of the amount of PTH peptide and total amount of
PTH analogs." Furthermore, "1.0% or less" and "5.0% or less" mean
when absolutely no PTH analogs are contained in the freeze-dried
preparation containing PTH peptide of the present invention or when
that % or less is contained.
[0223] Preferably, the freeze-dried preparation containing PTH
peptide of the present invention does not contain any more than
1.0% of at least one or more PTH analogs versus the "sum of the
amount of PTH peptide and total amount of PTH analogs," and more
preferably does not contain more than 1.0% of any PTH analog versus
the "sum of the amount of PTH peptide and total amount of PTH
analogs." In addition, when two analogs give a single peak on the
chromatogram, as mentioned above, the single peak is regarded as
one analog, and the analog regarded in this way is more preferably
not contained in an amount exceeding 1.0% versus the "sum of the
amount of PTH peptide and total amount of PTH analogs." The amount
of each PTH analog in the preparation is preferably "1.0% or less,"
but 0.9% or less, 0.8% or less, 0.7% or less, and 0.6% or less are
also preferred. Moreover, the total amount of PTH analogs is
preferably "5.0% or less," but 4.5% or less, 4.0% or less, 3.5% or
less, and 3.0% or less are also preferred.
[0224] An example of a suitable freeze-dried preparation containing
PTH peptide of the present invention appears below as Table 4.
(Furthermore, the "total amount of PTHs" in the table means the
"sum of the amount of PTH peptide and total amount of PTH
analogs.)
TABLE-US-00006 TABLE 4 Suitable freeze-dried preparation containing
PTH peptide of the present invention Content versus total amount of
PTHs PTH analog (%) (1) PTH-Met 8 [O]-Met 18 [O]-Trp 23 0.04% or
less, preferably 0.03% or [dioxidation] less (e.g. Human PTH
(1-34)-Met 8 [O]-Met 18 [O]-Trp 23 [dioxidation]) (3) Mixture
containing PTH-Met 8 0.11% or less, preferably 0.10% or [O]-Met 18
[O] and PTH-Met 8 [O]- less, most preferably 0.03% or less Trp 23
[dioxidation] (e.g., Mixture containing human PTH (1-34)-Met 8
[O]-Met 18 [O] and human PTH (1-34)-Met 8 [O]- Trp 23
[dioxidation]) (4) PTH-Met 18 [O]- Trp 23 0.26% or less, preferably
0.20% or [dioxidation] less, most preferably 0.06% or less (e.g.,
Human PTH (1-34)-Met 18 [O]- Trp 23 [dioxidation]) (6) PTH-Met 8
[O] 0.33% or less, preferably 0.30% or (e.g., Human PTH (1-34)-Met
8 [O]) less, most preferably 0.23% or less (7) PTH-Met 18 [O]
Arbitrary percentage selected from (e.g., Human PTH (1-34)-Met 18
[O]) 1.01-2.00% or less, preferably 1.00% or less, more preferably
0.50% or less, most preferably 0.36% or less (8) PTH-Trp 23
[dioxidation] 0.68% or less, preferably 0.50% or (e.g., Human PTH
(1-34)-Trp 23 less, most preferably 0.11% or less
[dioxidation])
[0225] To further explain the freeze-dried preparation containing
PTH peptide of the present invention, the freeze-dried preparation
containing PTH peptide of the present invention can contain various
additives. Examples of additives include sugars, amino acids,
sodium chloride, and the like. When sugars are used as additives,
mannitol, glucose, sorbitol, inositol, sucrose, maltose, lactose,
or trehalose is preferably added in an amount of 1 weight or more
(preferably 50-1000 weights) per weight of PTH peptide. When sugars
and sodium chloride are used as additives, sodium chloride is
preferably added in an amount of 1/1000-1/5 weight (preferably
1/100 to 1/10 weight) per weight of sugars.
[0226] (5) Container of the Freeze-Dried Preparation Containing PTH
Peptide of the Present Invention
[0227] The container used for the freeze-dried preparation
containing PTH peptide of the present invention is not particularly
restricted, but the preparation is preferably a freeze-dried
preparation containing PTH peptide housed in a stoppered glass
container. The material of the stopper is not particularly
restricted, but rubber is preferred. The stopper is preferably
washed, sterilized, and/or dried.
[0228] The freeze-dried preparation containing PTH peptide of the
present invention housed in a stoppered glass container is, for
example, a freeze-dried preparation containing PTH peptide housed
in a glass vial having a rubber stopper (glass vial preparation),
kit preparation comprising a freeze-dried preparation containing
PTH peptide housed in a glass vial having a rubber stopper and an
ampule sterilely filled with aqueous solution for dissolution, kit
preparation comprising a freeze-dried preparation containing PTH
peptide and prefilled syringe sterilely filled with aqueous
solution for dissolution, or glass double chamber preparation (two
chambers are present in one syringe, one chamber containing
freeze-dried preparation containing PTH peptide and the other
containing aqueous solution for dissolution). A glass vial
preparation is most preferred as the freeze-dried preparation
containing PTH peptide of the present invention. Examples of the
material of the rubber stopper include chlorinated butyl rubber,
normal butyl rubber, butadiene rubber, isoprene rubber, silicone
rubber, elastomer, and the like. Silicate glass is preferred as the
glass.
[0229] (6) Production of a Freeze-Dried Preparation Containing PTH
Peptide
[0230] A freeze-dried preparation is produced by a process
typically including any or all of the following steps, depending on
its use. Unless particularly stated otherwise, the freeze-dried
preparation containing PTH peptide of the present invention can
also be produced according to the following steps. Specifically the
production scheme of the freeze-dried preparation containing PTH
peptide of the present invention includes at least an active
ingredient-containing solution preparation step and a freeze drying
step explained below. It usually includes an active
ingredient-containing solution preparation step, loading step, and
freeze drying step, and preferably includes an active
ingredient-containing solution preparation step, aseptic filtration
and drug solution dispensing step, loading step, freeze drying
step, and packaging step.
[0231] 1) Active Ingredient Solution Preparation Step
[0232] This step dissolves a bulk compound of the active ingredient
and various additives as needed in a solvent (e.g., water for
injection). Adjustment of the pH of the solution, adjustment of the
volume of the solution, and the like may be performed as needed.
The time necessary for this step is not particularly restricted as
long as it is within the acceptable range for industrial
production, but it may be 0.5-5 hours, usually about 1-3 hours.
[0233] When the PTH peptide of the present invention is the active
ingredient, it is preferable to dissolve the bulk PTH peptide in
advance and add it to a solution in which the various additives
have been dissolved. Examples of additives include excipients,
stabilizers, dissolution auxiliaries, antioxidants, analgesics,
isotonifying agents, pH regulators, and preservatives.
[0234] 2) Aseptic Filtration and Drug Solution Dispensing Step
[0235] This step includes aseptic filtration of the active
ingredient-containing solution prepared in the above step and
filling a container suited to the performance of the freeze drying
step explained below with this aseptic filtered solution (drug
solution).
[0236] In a typical step, aseptic filtration is carried out using a
filter. Various commercial products can be used as the filter for
aseptic filtration. The pore size of the filter is preferably 0.2
.mu.m or less or 0.22 .mu.m or less. Specific equipment and the
like for performing aseptic filtration is well known to those
skilled in the art. Such aseptic filtration makes it possible to
prepare a drug solution for producing a freeze-dried preparation to
be utilized as a pharmaceutical.
[0237] Typical drug solution filling in this step is also well
known to those skilled in the art. Usually, individual containers
are filled directly with drug solution after aseptic filtration of
the solution of the active ingredient. Alternatively, a large
amount of solution may be aseptically filtered at once and
subsequently dispensed into containers suited to use in the
following step. An example of these containers is a glass vial that
can be stoppered by a rubber stopper or the like. The use of such
glass vials is advantageous in the production of a preparation in a
glass vial.
[0238] The time necessary for this step is also not particularly
restricted as long as it is within the acceptable range for
industrial production, but it may be 0.5-2 hours, usually 0.5-1
hour, as the filtration step and 3-10 hours, usually 6-10 hours, as
the filling step.
[0239] Furthermore, when the freeze-dried preparation containing
PTH peptide of the present invention is made into a preparation in
a glass vial, one glass vial can be filled, for example, with about
1 g (preferably 0.3-3 g, more preferably 0.5-0.6 g) of aseptically
filtered solution containing PTH peptide.
[0240] 3) Loading Step
[0241] The term loading step here means a series of steps whereby
the filled containers prepared as described above are transported
(transferred) to the freeze-drying means used in the next step and
loaded and placed in that means.
[0242] The stoppers of the filled containers are usually open or
partially open to dry by vacuum the filled solution frozen in a
subsequent step in the production of a freeze-dried preparation. An
open stopper means that the stopper is completely open, and a
partially open stopper means that the stopper is not open but not
closed. This makes it possible to vacuum dry the drug solution in
the container after freezing. For example, when the product is a
preparation in a glass vial, a partially open stoppered state as
above is created by stoppering the filled vial partially using a
rubber stopper after filling the glass vial by aseptically filtered
solution (drug solution). When the freeze-dried preparation
containing PTH peptide of the present invention is made as a
preparation in a glass vial or the like, a step to provide partial
stoppering in this way is also included in this loading step.
[0243] A freeze drying means is means making it possible to dry the
frozen solution under vacuum. Means for industrial production is
preferably also provided with an adequate cooling function to
freeze the solution or preferably provided with a function to
properly heat the material to be freeze dried during this treatment
to accelerate freeze drying. Since the material to be freeze dried
is loaded into a chamber in a typical freeze-drying means suited to
industrial production, this means has a large door (also referred
to as "large door" hereinafter) corresponding to basically its
entire front surface. A typical freeze-drying means is a
freeze-drying chamber (also called a "freeze dryer"), and many
forms of these are marketed.
[0244] To explain this step by way of example, taking a preparation
in glass vials as an example of the preparation to be produced, a
process whereby glass vials filled with drug solution obtained in
the above step "2)" are partially stoppered and transported to the
freeze-drying means and each vial is loaded sequentially or a
quantity unit is loaded together at one time into the freeze-drying
chamber and placed therein corresponds to this step. Furthermore,
when it is noted here that each vial is "loaded sequentially,"
depending on the layout of the pharmaceutical production facility,
this can mean that each vial is filled one after another
continuously by the above drug solution dispensing step, and each
vial is then sequentially partially stoppered and transferred
(transported) to the freeze-drying means. Usually, however, since
the process moves to the next freeze-drying step after all of the
vials that can be treated at once by the freeze-drying means have
been loaded into the freeze-drying means, each of the vials
transported as described above is introduced one after another
(that is, "sequential loading") and placed into the freeze-drying
means until reaching the quantity that can be treated at once.
However, in the case of such "sequential loading," the "loading
step" of the present invention means a step that begins with a
certain (first) vial after the drug solution dispensing step has
been completed until the final vial to be freeze dried together
with this (first) vial (that is, at once) has been loaded and
placed in the freeze-drying means.
[0245] Loading the vials "in a quantity unit together at one time"
can mean, for example, that there are multiple trays in the
freeze-drying chamber and multiple drug solution-filled vials are
placed together on each tray when placing them in the freeze-drying
chamber, and sometimes these trays can be moved up and down for the
sake of convenience when loading the drug solution-filled vials. In
this case as well, the "loading step" of the present invention
means a step that begins with a certain (first) vial after the drug
solution dispensing step has been completed until the final vial to
be freeze dried together with this (first) vial has been loaded and
placed in the freeze-drying means. In any case, the drug
solution-filled vials are left in a partially open state in this
step until the subsequent freeze drying step begins and may be
exposed to the air environment within a pharmaceutical production
facility explained below.
[0246] The time necessary for this step also is not particularly
restricted as long as it is within the range acceptable for
industrial production, but is 3-10 hours, usually about 6-10
hours.
[0247] 4) Freeze Drying Step
[0248] This is a step for sublimating water from the frozen
material to be dried under reduced pressure by the above freeze
drying means. When a freeze-dried preparation is produced in glass
vials, the vials can be placed in an open or partially open state
under reduced pressure (for example, with the vials partially
stoppered), and sealed at the end of freeze drying after the space
in the vial has been purged by nitrogen.
[0249] The time necessary for this step varies depending on the
capabilities of the freeze drying means, amount of substance to be
freeze dried, and the like and should be within the range
acceptable for industrial production. It is usually about 24-72
hours.
[0250] 5) Closing Step
[0251] This step can be included when producing a freeze-dried
preparation in a glass vial. Specifically, it is a step whereby the
freeze-dried glass vials obtained in the above step "4)" are closed
by an aluminum cap by a press-type capping machine, or the
like.
[0252] 6) Packaging Step
[0253] This is a step that attaches a label to the preparation and
packages it in a paper box or the like.
[0254] When a freeze-dried preparation is produced as a
pharmaceutical, the production facility must be a facility that
meets the pharmaceutical GMP. This facility has drug solution
preparation equipment, aseptic filtration equipment, and freeze
drying equipment (means) and, in addition to these, water for
injection production equipment, vial filling and stoppering
equipment, capping machines, labelers, and the like to implement
the steps explained above.
[0255] When a freeze-dried preparation is produced as a
pharmaceutical, all of the above steps 1)-6) or at least from the
end of the aseptic filtration step to the beginning of the freeze
drying step should be carried out in air environments within a
pharmaceutical production facility. That is, the air environment of
the pharmaceutical production facility differs from the simple
outside air environment. Specifically, it is required that the air
environment inside a facility for production of a sterile injection
(pharmaceutical) be "a critical area of high cleanliness (the
content of suspended microparticles 0.5 .mu.m or greater in size
per m.sup.3 of air is to be 3520 or fewer during both work and
non-work times)." This air quality corresponds to grade A (termed
class 100 or ISO 5) according to current, commonly-used domestic
and international air quality standards.
[0256] The air environment inside the facility that produces a
freeze-dried preparation containing PTH peptide of the present
invention should be at least equivalent to the air environment
inside the above sterile injection production facility, more
preferably an environment that maintains a unidirectional flow
downward from above of clean air that has passed through HEPA
filters capable of trapping particles of 0.3 .mu.m in size at an
efficiency of 99.97% or greater. The air flow speed is preferably
0.2-1.0 m/s at a location 20 cm below the HEPA filter and 0.1-0.8
m/s at the location where the production work is carried out, more
preferably 0.4-0.7 m/s at a location 20 cm below the HEPA filter
and 0.3-0.5 m/s at the location where the production work is
carried out.
[0257] To create a more sterile air environment inside a
pharmaceutical production facility, bacteria suspended in the air
and bacteria adhered to machinery, walls, floors, and other such
installations are sterilized using ozone or formaldehyde or
chemicals having oxidizing capability, such as hydrogen peroxide,
peracetic acid, chlorine dioxide, glutaraldehyde, and the like, as
disinfectants. The residual formaldehyde concentration after
fumigation and sterilization by formaldehyde should usually be kept
to 0.1 ppm or lower, preferably 0.08 ppm or lower. Furthermore, in
terms of ozone, ozone is usually present even in the outside air in
a concentration of 0.001-0.02 ppm as an average daily value.
Concentrations of approximately 0.02-0.1 ppm are also sometimes
present temporarily depending on the time, location, and
season.
[0258] As one embodiment of the present invention, a method for
producing a freeze-dried preparation containing PTH peptide is
characterized in that exposure of the solution containing PTH
peptide to the air environment within a pharmaceutical production
facility is controlled during the process of the course when a
substantial time is required under the air environment within a
pharmaceutical production facility as described above at the
beginning of the step for preparing a solution of PTH peptide
(active ingredient), especially from the end of the drug solution
dispensing step to the beginning of the drug solution freeze drying
step (that is, loading step).
[0259] In the present invention, "exposure to air environments
within a pharmaceutical production facility is controlled" and
"control of exposure to air environments within a pharmaceutical
production facility" mean both that at least one or more of the PTH
peptide bulk drug, solution containing PTH peptide, and PTH peptide
freeze-dried preparation have absolutely no contact with air
environments within a pharmaceutical production facility and that
this contact is substantially (e.g., time and level of contact)
restricted. For example, in the case of an environment in which
clean air that has passed through HEPA filters as described above
is maintained as a unidirectional flow in a direction downward from
above (referred to hereinafter as "flowing air"), this includes
provision of a means to control the time in contact with this
flowing air and the contacted air flow rate. Specific examples
appear below.
[0260] (A) Means to Control Contact of the Solution Containing PTH
Peptide with Flowing Air
[0261] As described above, the present inventors discovered that
the generation of impurities (PTH analogs) in solution containing
PTH peptide can be controlled by providing means for controlling
contact of the solution containing PTH peptide with flowing air.
Since flow of the air within the facility is maintained in an
ordinary pharmaceutical production facility, it can be deduced that
a large amount of air that pours down as air flow comes into
contact with the solution containing PTH peptide and that gaseous
substances having oxidizing capability (ozone and the like)
contained in this air flow increase the PTH analogs in the solution
by causing reactions and the like with the PTH peptide in the
solution.
[0262] The means for controlling contact of the solution containing
PTH peptide with flowing air in the present invention is not
particularly restricted. Examples include a means for controlling
the fluidity and flow of the air in the vicinity of the solution
containing PTH peptide and a means for purging the vicinity of the
solution containing PTH peptide with an inert gas.
[0263] It was also discovered that it is advantageous to provide
means for controlling contact of the solution containing PTH
peptide with flowing air in the loading step into the freeze-drying
chamber.
[0264] To explain the above embodiment based on a non-limiting
example, an ordinary freeze-drying chamber has a door in the front
surface for loading the containers filled with the solution to be
freeze dried. This door is often a door (large door) that can cover
the entire front surface of the freeze-drying chamber. The present
invention, however, at a part of the large door, additionally
provides a small door of a size roughly corresponding to one of the
trays placed in the freeze-drying chamber (with the filled
containers to be freeze dried disposed on top of it), and a
freeze-drying chamber having a small door that can be opened and
closed easily to load the containers is preferred.
[0265] A more preferred example is the above freeze-drying chamber
with a small door having sub-doors that can be opened and closed
easily provided with openings for loading (also referred to
hereinafter as "small door openings") to create small door units to
open when loading and unloading the containers filled with the
material to be freeze dried into the freeze-drying chamber and a
means to open the sub-door only during container loading without
leaving it constantly open and to close it quickly after loading.
These sub-doors are provided divided into 2-5 levels among the
zones corresponding to the small door openings so that only the
necessary unit may be opened for loading. Sub-doors that make it
possible to open only the necessary location for loading the
containers are preferred, and division into 2-3 levels is
preferred. Examples of sub-doors that can opened and closed easily
include a sub-door that provides a hinge (hinge) at the top of the
sub-door and is installed in the small door opening, a sub-door
that slides to the right and left, a sub-door that slides up and
down, and the like.
[0266] Preferred examples of other means of controlling contact of
the solution containing PTH peptide and the like with flowing air
include sealing the equipment for preparation (tank or container
and the like) after the PTH peptide has been dissolved in the
solvent in the solution containing PTH peptide preparation step or
purging the interior of the container used for preparation by an
inert gas during preparation.
[0267] The inside of the preparation equipment (tank or container
and the like) is sometimes pressurized and the prepared solution
containing PTH peptide is passed through a sterile filter and fed
to the container or tank for dispensing in the aseptic filtration
and drug solution dispensing step. Another preferred example of a
means for controlling contact of the solution containing PTH
peptide with flowing air is to use an inert gas as the gas for
pressurization in this case.
[0268] Another preferred example of a means for controlling contact
of the solution containing PTH peptide with flowing air is to purge
the air inside the drug solution dispensing equipment (tank or
container and the like) with an inert gas in advance in the aseptic
filtration-drug solution dispensing step and to purge the glass
container to be filled with the solution containing PTH peptide
with an inert gas in advance.
[0269] Alternatively, another preferred example of means for
controlling contact of the solution containing PTH peptide with
flowing air is to purge the space inside the glass container (part
containing air and no drug solution) filled with the solution
containing PTH peptide with an inert gas in the aseptic
filtration-drug solution dispensing step.
[0270] Furthermore, the glass containers filled with the solution
containing PTH peptide are sometimes transported from the aseptic
filtration-drug solution dispensing equipment to near the
freeze-drying chamber during the time after the end of the aseptic
filtration-drug solution dispensing step up to loading of the
solution containing PTH peptide housed in the open or partially
open glass containers into the freeze drying means. Placing the
environment during transport under an inert gas flow can also be
given as a preferred example of a means for controlling contact of
the solution containing PTH peptide with flowing air in such
cases.
[0271] Alternatively, a flap or airflow-adjusting cover (FIG. 17)
that can change the flow of the flowing air from the opening into
the chamber can be installed to control the influx of flowing air
from the small opening of the freeze-drying chamber into the
freeze-drying chamber as a means for controlling contact of the
solution containing PTH peptide with flowing air. The shape of this
flap or airflow-adjusting cover can be selected as is appropriate
to the size of the freeze dryer and small door opening. It may be
made of a vinyl sheet, metal, resin, or the like. Furthermore,
controlling the influx of flowing air from the small door opening
of the freeze-drying chamber into the freeze-drying chamber means
that the influx of flowing air is controlled to the point that
contact between the solution containing PTH peptide and flowing air
is substantially controlled, preferably controlled so that the
inflow speed of flowing air from the small door opening is 0.2 m/s
or less, more preferably 0.1 m/s or less, and most preferably 0.0
m/s or less. This control can be achieved by proper placement of a
flap or airflow-adjusting cover or the like near the small
door.
[0272] In addition to purging means using inert gas as described
above, the means for purging the vicinity of the solution
containing PTH peptide with inert gas can be means for purging the
air inside the freeze-drying chamber used in the freeze drying step
using an inert gas or means for causing an inert gas to flow from
the loading port into the freeze-drying chamber when loading the
containers of solution containing PTH peptide into the
freeze-drying chamber used in the freeze drying step. The flow rate
of the inert gas during influx is preferably 0.1-5 Nm.sup.3/min,
more preferably 0.2-3 Nm.sup.3/min, and most preferably 0.3-1
Nm.sup.3/min. Examples of the inert gas in purging by an inert gas
include nitrogen and argon; nitrogen can be given as a preferred
example.
[0273] (B) Step that Provides Means for Controlling Contact of the
Solution Containing PTH Peptide with Flowing Air
[0274] The "means for controlling contact of the solution
containing PTH peptide with flowing air" of (A) above can be
provided in all or some steps included from the beginning of the
step for preparing a solution containing PTH peptide to the
beginning of the freeze drying step of this solution, and may be
provided from the beginning of the step for preparing a solution
containing PTH peptide. When the method for producing a
freeze-dried preparation containing PTH peptide as a pharmaceutical
of the present invention includes a step for preparing a solution
containing PTH peptide, a step for loading this solution housed in
open or partially open glass containers into a freeze-drying
chamber, and a freeze drying step, the means of (A) above can be
provided in some or all of the step for loading this solution
housed in open or partially open glass containers into a
freeze-drying chamber.
[0275] (C) Duration of the Step that Provides Means for Controlling
Contact of the Solution Containing PTH Peptide with Flowing Air
[0276] The duration of the step that provides "means for
controlling contact of the solution containing PTH peptide with
flowing air" of (A) above can have, for example, as the lower
limit, one hour or more, preferably three hours or more, and more
preferably six hours or more, and, as the upper limit, 20 hours or
less, preferably 12 hours or less, more preferably 10 hours or
less, and most preferably nine hours or less. Examples of the
duration of the step that provides a means of (A) above include
1-20 hours, preferably 3-12 hours, more preferably 6-10 hours, and
most preferably 6-9 hours.
[0277] (7) Use of the Freeze-Dried Preparation Containing PTH
Peptide
[0278] The freeze-dried preparation containing PTH peptide of the
present invention can contain a pharmaceutically effective amount
of PTH peptide and, for example, the freeze-dried preparation can
be dissolved in a suitable solvent at the time of use to make an
injection and used in the treatment of osteoporosis.
[0279] (8) Method for Controlling the Production of PTH Analogs in
Solution Containing PTH Peptide
[0280] The method for controlling the production of PTH analogs of
the present invention is a method that provides means for
controlling contact of at least one of the PTH peptide bulk drug,
solution containing PTH peptide, and freeze-dried preparation
containing PTH peptide with substances having oxidizing capability,
especially with air containing these substances. A preferred
example of a method for controlling the production of PTH analogs
is to provide a means for purging air in contact with solution
containing PTH peptide by an inert gas (preferably nitrogen). A
more preferred example is a method for controlling the production
of any one or more PTH analogs among the aforementioned analogs 1
to 11 and analogs 1' to 11' by means for controlling contact of the
solution containing PTH peptide with flowing air or means for
purging the air in contact with the solution containing PTH peptide
by an inert gas (preferably nitrogen).
[0281] These production control methods can be implemented in
freeze-dried preparation production facilities in air environments
within pharmaceutical production facilities as described above. For
example, their production in the solution can be controlled by
means for controlling contact of the solution with flowing air
during the course of the process for a predetermined time or longer
from the beginning of the step for preparing a solution containing
PTH peptide to the beginning of the freeze drying step of this
solution. Preferred embodiments of this means are the same as the
preferred embodiments of the corresponding methods for production a
freeze-dried preparation containing PTH peptide of the present
invention.
EXAMPLES
[0282] The present invention is explained more concretely below
through examples, reference examples, and test examples without
limiting the scope of the invention.
Example 1
[0283] Approximately 18 kg of approximately 25.degree. C. water for
injection was placed in a 50 L stainless steel container. A
quantity of 540 g of sucrose and 27 g of sodium chloride were
weighed out into the container and dissolved. Next, 3541 mg of
human PTH (1-34) as an acetate (lot A; 860 mg, lot B; 2591 mg, lot
C; 90 mg) was added and dissolved. A PTH peptide-containing aqueous
solution was then obtained by adding water for injection and
correcting the weight to 27 kg. The PTH peptide-containing aqueous
solution obtained was aseptically filtered using a filter while
pressurizing by nitrogen and fed to a 50 L stainless steel filling
tank previously filled with nitrogen. Within a zone having a grade
A (air speed approximately 0.2-0.4 m/s) environment within a
pharmaceutical production facility, washed and dried glass vials
were filled with 0.56 g of this aseptically filtered PTH
peptide-containing aqueous solution, and partially open vials were
obtained using washed and dried rubber stoppers. Approximately 1000
vials each were lined up on stainless steel trays, and the trays
were then transferred to in front of a freeze-drying chamber
manufactured by Ulvac (model: DFB, tray areas: 24 m.sup.2)
previously filled with nitrogen in a grade A zone. While purging
the inside of the freeze-drying chamber by nitrogen, a sub-door
that matched the width of the trays (a door equivalent to the
sub-door of FIG. 16 fitted in-house to the above commercial
freeze-drying chamber), provided in the opening when the small door
of the above freeze-drying chamber was open, was opened, and the
sub-door was shut quickly after the tray had been loaded into the
freeze-drying chamber. The same procedure was repeated, and the
partially open vials were loaded into the freeze-drying chamber
over approximately nine hours. The solution containing PTH was
frozen and freeze dried to sublimate off the water under reduced
pressure. After the interiors of the glass vials were purged with
nitrogen after drying had been completed, the vials were sealed
with the rubber stoppers, and capped with aluminum caps, resulting
in a freeze-dried preparation containing PTH peptide.
Example 2
[0284] Approximately 10 kg of approximately 25.degree. C. water for
injection was placed in a 20 L stainless steel container. A
quantity of 280 g of sucrose and 14 g of sodium chloride were
weighed out into the container and dissolved. Next, the weight was
corrected to 14 kg by adding water for injection, and an additive
solution was prepared. A PTH peptide-containing aqueous solution
was obtained by weighing out 1780 mg (lot D) of human PTH (1-34) as
an acetate and dissolving it in 13 kg of the additive solution. The
PTH peptide-containing aqueous solution obtained was aseptically
filtered using a filter while pressurizing by nitrogen and fed to a
50 L stainless steel filling tank previously filled with nitrogen.
Within a zone having a grade A (air speed approximately 0.2-0.4
m/s) environment within a pharmaceutical production facility,
washed and dried glass vials were filled with 0.56 g of this
aseptically filtered PTH peptide-containing aqueous solution, and
partially open vials were obtained using washed and dried rubber
stoppers. Approximately 1000 vials each were lined up on stainless
steel trays, and the trays were then transferred to in front of a
freeze-drying chamber manufactured by Ulvac (model: DFB, tray
areas: 24 m.sup.2) previously filled with nitrogen in a grade A
zone. While purging the inside of the freeze-drying chamber with
nitrogen, a sub-door that matched the width of the trays (a door
equivalent to the sub-door of FIG. 16 fitted in-house to the above
commercial freeze-drying chamber), provided in the opening when the
small door of the above freeze-drying chamber was opened, and the
sub-door was closed quickly after the tray had been loaded into the
freeze-drying chamber. The same procedure was repeated, and the
partially open vials were loaded into the freeze-drying chamber
over approximately six hours. The solution containing PTH peptide
was frozen and freeze dried to sublimate off the water under
reduced pressure. After the interiors of the glass vials were
purged with nitrogen after drying had been completed, the vials
were sealed with the rubber stoppers, capped with aluminum caps,
and a freeze-dried preparation containing PTH peptide was
obtained.
Example 3
[0285] Approximately 19 kg of approximately 25.degree. C. water for
injection was placed in a 30 L stainless steel container. A
quantity of 460 g of sucrose and 23 g of sodium chloride were
weighed out into the container and dissolved. Next, the weight was
corrected to 23 kg by adding water for injection, and a placebo
solution was prepared. A PTH peptide-containing aqueous solution
was obtained by weighing out 2979 mg (lot D) of human PTH (1-34) as
an acetate and dissolving it in 22 kg of the placebo solution. The
PTH peptide-containing aqueous solution obtained was aseptically
filtered using a filter while pressurizing by nitrogen and fed to a
stainless steel filling tank previously filled with nitrogen.
Within a zone having a grade A (air speed approximately 0.2-0.4
m/s) environment within a pharmaceutical production facility,
washed and dried glass vials were filled with 0.56 g of this
aseptically filtered PTH peptide-containing aqueous solution, and
partially open vials were obtained using washed and dried rubber
stoppers. The partially open vials were transferred to a grade A
zone, and all of the vials were loaded over approximately six hours
into a freeze-drying chamber manufactured by Ulvac (model: DFB,
tray areas: 22 m.sup.2) previously filled with nitrogen made so
that air flow from the opening of the small door does not enter the
freeze-drying chamber using a vinyl sheet (a sheet equivalent to
the airflow-adjusting cover of FIG. 17 fitted in-house to the above
commercial freeze-drying chamber) capable of changing the flow of
the flowing air to the opposite direction from the opening of the
small door of the freeze-drying chamber. Furthermore, this vinyl
sheet capable of changing the flow of the flowing air was also
fitted in-house to the above commercial freeze-drying chamber; a
vinyl sheet was stretched obliquely downward from the upper part of
the opening of the small door, and flowing air was prevented from
entering from the opening by changing the direction of the flowing
air flowing from above to below. The solution containing PTH
peptide was frozen and freeze dried to sublimate off the water
under reduced pressure. After the interiors of the glass vials were
purged with nitrogen once drying had been completed, the vials were
sealed with the rubber stoppers, capped with aluminum caps, and a
freeze-dried preparation containing PTH peptide was obtained.
Example 4
[0286] Approximately 18 kg of approximately 25.degree. C. water for
injection was placed in a 50 L stainless steel container. A
quantity of 540 g of sucrose and 27 g of sodium chloride were
weighed out into the container and dissolved. Next, 3525 mg of
human PTH (1-34) as an acetate (lot C; 1880 mg, lot E; 1645 mg) was
added and dissolved. A PTH peptide-containing aqueous solution was
then obtained by adding water for injection and correcting the
weight to 27 kg. The PTH peptide-containing aqueous solution
obtained was aseptically filtered using a filter while pressurizing
by nitrogen and fed to a 50 L stainless steel filling tank
previously filled with nitrogen. Within a zone having a grade A
(air speed approximately 0.2-0.4 m/s) environment and a formalin
concentration brought to 0.08 ppm or lower within a pharmaceutical
production facility, washed and dried glass vials were filled with
0.56 g of this PTH peptide-containing aqueous solution, and
partially open vials were obtained using washed and dried rubber
stoppers. Approximately 1000 vials each were lined up on stainless
steel trays, and the trays were then transferred to in front of a
freeze-drying chamber manufactured by Ulvac (model: DFB, tray
areas: 24 m.sup.2) previously filled with nitrogen in a grade A
zone. While purging the inside of the freeze-drying chamber by
nitrogen, a sub-door that matched the width of the trays (a door
equivalent to the sub-door of FIG. 16 fitted in-house to the above
commercial freeze-drying chamber), provided in the opening when the
small door of the above freeze-drying chamber was open, was opened,
and the sub-door was closed quickly after the tray had been loaded
into the freeze-drying chamber. The same step was repeated, and the
partially open vials were loaded into the freeze-drying chamber
over approximately eight hours. The solution containing PTH peptide
was frozen and freeze dried to sublimate off the water under
reduced pressure. After the interiors of the glass vials were
purged with nitrogen once drying had been completed, the vials were
sealed with the rubber stoppers, capped by aluminum caps, and a
freeze-dried preparation containing PTH peptide was obtained.
Example 5
[0287] Approximately 18 kg of approximately 25.degree. C. water for
injection was placed in a 50 L stainless steel container. A
quantity of 540 g of sucrose and 27 g of sodium chloride were
weighed out into the container and dissolved. Next, 3566 mg of
human PTH (1-34) as an acetate (lot H) was added and dissolved. A
PTH peptide-containing aqueous solution was then obtained by adding
water for injection and correcting the weight to 27 kg. The PTH
peptide-containing aqueous solution obtained was aseptically
filtered using a filter while pressurizing by nitrogen and fed to a
50 L stainless steel filling tank previously filled with nitrogen.
Within a zone having a grade A (air speed approximately 0.2-0.4
m/s) environment and a formalin concentration brought to 0.08 ppm
or lower within a pharmaceutical production facility, washed and
dried glass vials were filled with 0.56 g of this PTH
peptide-containing aqueous solution, and partially open vials were
obtained using washed and dried rubber stoppers. Approximately 1000
vials each were lined up on stainless steel trays, and the trays
were then transferred to in front of a freeze-drying chamber
manufactured by Ulvac (model: DFB, tray areas: 24 m.sup.2)
previously filled with nitrogen in a grade A zone. While purging
the inside of the freeze-drying chamber with nitrogen, a sub-door
that matched the width of the trays (a door equivalent to the
sub-door of FIG. 16 fitted in-house to the above commercial
freeze-drying chamber), provided in the opening when the small door
of the above freeze-drying chamber was open, was opened, and the
sub-door was closed quickly after the tray had been loaded into the
freeze-drying chamber. The same step was repeated, and the
partially open vials were loaded into the freeze-drying chamber
over approximately seven hours. The solution containing PTH peptide
was frozen and freeze dried to sublimate off the water under
reduced pressure. After the interiors of the glass vials were
purged with nitrogen once drying had been completed, the vials were
sealed with the rubber stoppers, capped by aluminum caps, and a
freeze-dried preparation containing PTH peptide was obtained.
Comparative Example 1
[0288] Approximately 5000 g of approximately 25.degree. C. water
for injection was placed in a 10 L stainless steel container. A
quantity of 120 g of sucrose and 6 g of sodium chloride were
weighed out into the container and dissolved. Next, 909 mg of human
PTH (1-34) as an acetate (lot F; 335 mg, lot G; 574 mg) was added
and dissolved. A PTH peptide-containing aqueous solution was then
obtained by adding water for injection and correcting the weight to
6000 g. The PTH peptide-containing aqueous solution obtained was
aseptically filtered using a filter while pressurizing by nitrogen
and fed to a stainless steel filling tank. Within a zone having a
grade A (air speed approximately 0.2-0.4 m/s) environment within a
pharmaceutical production facility, washed and dried vials were
filled with 0.56 g of this PTH peptide-containing aqueous solution,
and partially open vials were obtained using washed and dried
rubber stoppers. The partially open vials were transferred to a
grade A zone, and all were sequentially loaded over approximately
four hours into a freeze-drying chamber manufactured by Ulvac
(model: DFB, tray areas: 22 m.sup.2) having a small door. The
solution containing PTH peptide was frozen and freeze dried to
sublimate off the water under reduced pressure. After the interiors
of the glass vials were purged with nitrogen once drying had been
completed, the vials were sealed with the rubber stoppers, capped
with aluminum caps, and a freeze-dried preparation containing PTH
peptide was obtained.
Comparative Example 2
[0289] Approximately 2000 g of approximately 25.degree. C. water
for injection was placed in a 10 L stainless steel container.
Quantities of 100 g of sucrose and 5 g of sodium chloride were
weighed out into the container and dissolved. Next, 515 mg of human
PTH (1-34) as an acetate (lot D) was added and dissolved. A PTH
peptide-containing aqueous solution was then obtained by adding
water for injection and correcting the weight to 4000 g. The PTH
peptide-containing aqueous solution obtained was aseptically
filtered using a filter while pressurizing by nitrogen and fed to a
5 L stainless steel filling tank. Within a zone having a grade A
(air speed approximately 0.2-0.4 m/s) environment within a
pharmaceutical production facility, washed and dried vials were
filled with 0.56 g of this PTH peptide-containing aqueous solution,
and partially open vials were obtained using washed and dried
rubber stoppers. The partially open vials were transferred to a
grade A zone, and all were sequentially loaded over approximately
three hours into a freeze-drying chamber having a small door (Kyowa
Vacuum Engineering Co., Ltd. (model: RL, tray area: 9 m.sup.2)).
The solution containing PTH peptide was frozen and freeze dried to
sublimate off the water under reduced pressure. After the interiors
of the glass vials were purged with nitrogen once drying had been
completed, the vials were sealed with the rubber stoppers, capped
with aluminum caps, and a freeze-dried preparation containing PTH
peptide was obtained.
Test Example 1
[0290] The area percentage method using HPLC is a simple method for
assessing the purity of a freeze-dried preparation containing PTH
peptide and the amount of analogs. A solution obtained by weighing
out 0.25 g of benzalkonium chloride and bringing it to 50 mL by
adding 50 mM sulfate buffer (pH 2.3) serves as the loading buffer.
Each preparation of the examples and comparative examples is
dissolved by 1 mL of physiological saline solution, and a 9:1
mixture of this solution and the loading buffer serves as the
sample solution. A quantity of 100 .mu.L of sample solution is
tested by HPLC under the following conditions. Furthermore,
benzalkonium chloride was used to prevent the peptide that is the
target of measurement from attaching to the instrument and the
like.
[0291] <Test Conditions>
[0292] Detector: Ultraviolet absorptiometer (measurement
wavelength: 214 nm)
[0293] Column: Stainless steel tube 150 mm long having an inner
diameter of 4.6 mm packed with 3.5 .mu.m of octadecylsilylated
silica gel
[0294] Column temperature: Constant temperature near 40.degree.
C.
[0295] Mobile phase: Mobile phase A: Dissolve 28.4 g of anhydrous
sodium sulfate in 900 mL of water and bring to 1000 mL by adding
water after adding phosphoric acid to adjust the pH to 2.3. Add 100
mL of acetonitrile to this 900 mL of liquid.
[0296] Mobile phase B: Dissolve 28.4 g of anhydrous sodium sulfate
in 900 mL of water and bring to 1000 mL by adding water after
adding phosphoric acid to adjust the pH to 2.3 Add 500 mL of
acetonitrile to 500 mL of this liquid.
[0297] Mobile phase feed: Concentration gradient control is
provided by varying the mixture ratio of mobile phase A and mobile
phase B as follows.
[0298] Time after injection:
TABLE-US-00007 TABLE 5 Concentration gradient control Time after
sample injection (min) Mobile phase A (vol %) Mobile phase B (vol
%) 0-5 100.fwdarw.65 0.fwdarw.35 5-35 65.fwdarw.60 35.fwdarw.40
35-45 60.fwdarw.0 40.fwdarw.100
[0299] Flow rate: 1.0 mL/min
[0300] Sample temperature: Constant temperature near 5.degree.
C.
[0301] Detection time: 45 minutes after injection of the sample
solution. However, this is from the back of the solvent peak.
[0302] Calculation method: The amount of each PTH analog and the
total amount thereof was determined by performing liquid
chromatography under the above conditions, measuring each peak area
by automatic integration, and performing calculation using Formulas
1 and 2. Furthermore, the total peak area was the sum total of the
area of all peaks detected by performing liquid chromatography
under the above conditions. In other words, the total peak area
shows the sum total of PTH peptide and all PTH analogs in the
preparation.
Amount of each PTH analog (%)=(peak area of each analog/total peak
area).times.100 Formula 1:
Total amount of PTH analogs (%)=(sum total of peak areas of each
analog/total peak area).times.100 Formula 2:
[0303] <Results>
[0304] Table 6 shows the results obtained by assessing the amount
of analogs of the human PTH (1-34) (bulk drug) used in the
examples. FIG. 1 shows an HPLC chart. Table 7 shows the results
obtained by assessing the purity of the freeze-dried preparations
containing PTH peptide and the amount of analogs in the test
example. FIG. 2 shows an HPLC chart of Example 1, and FIG. 3 shows
an HPLC chart of Comparative Example 1. The structure of each
analog in Table 6 was obtained by estimation using Test Example 2
below.
TABLE-US-00008 TABLE 6 Amount of each analog (%) in human PTH
(1-34) used as a bulk drug D Use in examples and comparative A B C
Ex. 2, E F G H examples Ex. 1 Ex. 1 Ex. 1, 4 CEx 2 Ex. 4 CEx 1 CEx
1 Ex. 5 (1) Human PTH (1-34)-Met 8 [O]- ND ND ND ND ND ND ND ND Met
18 [O]-Trp 23 [dioxidation] (2) Human PTH (1-34)-Met 8 [O]- ND ND
ND ND ND ND ND ND Met 18 [O]-Trp 23 [dioxidation- formic acid
elimination] (3) Mixture containing human ND ND ND ND ND ND ND ND
PTH (1-34)-Met 8 [O]-Met 18 [O] and human PTH (1-34)-Met 8 [O]- Trp
23 [dioxidation] (4) Human PTH (1-34)-Met 18 ND ND ND ND ND ND ND
ND [O]-Trp 23 [dioxidation] (5) Human PTH (1-34)-Met 18 ND ND ND ND
ND ND ND ND [O]-Trp 23 [dioxidation-formic acid elimination] (6)
Human PTH (1-34)-Met 8 [O] 0.08 0.07 0.11 0.05 0.05 0.14 0.17 0.06
(7) Human PTH (1-34)-Met 18 [O] 0.13 0.09 0.16 0.06 0.07 0.21 0.23
0.09 (8) Human PTH (1-34)-Trp 23 ND ND ND ND ND ND ND ND
[dioxidation] (9) Mixture containing human ND ND ND ND ND ND ND ND
PTH (1-34)-Trp 23 [monoxidation] and human PTH (1-34)-Trp 23
[dioxidation- formic acid elimination] Ex.: Example; CEx.:
Comparative Example
TABLE-US-00009 TABLE 7 Total amount of analogs (total amount) and
amount of each analog (%) Ex. 1 Ex. 2 Ex. 3 Ex. 4 Ex. 5 CEx. 1 CEx.
2 Total amount (%) 0.88 0.76 1.51 0.95 0.72 6.27 3.09 (1) Human PTH
(1-34)-Met 8 [O]- 0.03 ND ND ND ND 0.10 0.05 Met 18 [O]-Trp 23
[dioxidation] (2) Human PTH (1-34)-Met 8 [O]- 0.02 ND ND ND ND 0.07
ND Met 18 [O]-Trp 23 [dioxidation- formic acid elimination] (3)
Mixture containing human PTH 0.03 ND ND 0.03 0.03 0.29 0.12
(1-34)-Met 8 [O]-Met 18 [O] and human PTH (1-34)-Met 8 [O]-Trp 23
[dioxidation] (4) Human PTH (1-34)-Met 18 [O]- 0.06 0.02 0.03 0.05
ND 0.69 0.27 Trp 23 [dioxidation] (5) Human PTH (1-34)-Met 18 [O]-
ND ND 0.03 ND ND 0.19 ND Trp 23 [dioxidation-formic acid
elimination] (6) Human PTH (1-34)-Met 8 [O] 0.14 0.09 0.24 0.15
0.09 0.93 0.34 (7) Human PTH (1-34)-Met 18 [O] 0.19 0.15 0.36 0.25
0.10 1.42 1.08 (8) Human PTH (1-34)-Trp 23 0.09 0.04 0.10 0.11 0.04
0.74 0.69 [dioxidation] (9) Mixture containing human PTH 0.05 ND
0.09 0.04 0.02 0.40 0.08 (1-34)-Trp 23 [monoxidation] and human PTH
(1-34)-Trp 23 [dioxidation-formic acid elimination] Ex.: Example;
CEx.: Comparative Example
Test Example 2
[0305] Human PTH (1-34) analogs were produced and fractionated, and
the fractions were analyzed to estimate the structures of each
analog obtained in Test Example 1.
[0306] (1) Production and Fractionation of Each Analog
[0307] A quantity of 4.00 g of sucrose and 0.20 g of sodium
chloride were weighed out and dissolved by adding water for
injection to make a placebo solution. Human PTH (1-34) was weighed
out exactly and dissolved by adding 100 mL of placebo solution to
make a reaction stock solution. An environment having an ozone
concentration of approximately 0.08 ppm by ozone concentration
meter was produced using an ozone generator and a blower (initial
wind speed approximately 7.2 m/s) to circulate the ozone and make
the concentration uniform in a tray having a 40 cm long.times.90 cm
wide.times.100 cm high glass door. Approximately 15 mL of the
reaction stock solution was dispensed into each 20 mL vial. A
stirrer was introduced into the vial, and degradation was carried
out while stirring by stirrer until the purity was approximately
20% (in other words, the total amount of human PTH (1-34) analogs
was 80%) by exposure (for about 20 hours) to an ozone atmosphere of
approximately 0.08 ppm. Furthermore, the purity was confirmed in
accordance with the test conditions of Test Example 1. The degraded
solution was freeze dried, and a solution dissolved by a suitable
amount of water for injection was taken as a forced-degradation
solution. The analogs were fractionated under the following
conditions using this solution.
[0308] <Test Conditions>
[0309] The test conditions other than the following were the same
as the test conditions in Test Example 1.
[0310] <Conditions Different from Test Example 1>
[0311] Column: Stainless steel tube 250 mm long having an inner
diameter of 9.4 mm packed with 5 .mu.m of octadecylsilylated silica
gel
[0312] Flow rate: 6.0 mL/min
[0313] Furthermore, no major differences in the chromatogram
pattern were observed despite the fact that the above conditions
differed from those of Test Example 1.
[0314] Nine analogs were fractionated under the above
chromatography conditions. They were desalted and concentrated, and
the freeze-dried products were dissolved in distilled water to
obtain each analog (undigested compound). Each analog (undigested
compound) and the forced-degradation solution were analyzed by HPLC
under the test conditions of Test Example 1, and the relative
retention time of each analog (undigested compound) was calculated,
taking the retention time of human PTH (1-34) in the
forced-degradation solution as 1.
[0315] <Results>
[0316] Basically complete agreement with the relative retention
time of each analog in the forced-degradation solution was
confirmed. Table 8 shows the results on the relative retention
times. FIG. 4 shows a chromatogram of the forced-degradation
solution. The elution times of the human PTH (1-34) peaks differ
slightly owing to the differing compositions of the charged
solutions in FIGS. 3 and 4, but the corresponding analogs of FIGS.
3 and 4 were assumed to be the same given that the elution patterns
and weight percent of each analog were the same on each chart.
Based on these results, the ozone exposure test here appeared to be
a test that substantially reproduces the PTH analog production
reactions triggered when solution containing PTH peptide is
produced in an air environment inside a pharmaceutical production
facility.
TABLE-US-00010 TABLE 8 Comparison of the relative retention time of
the relevant peaks of analogs (undigested compounds) and
forced-degradation solution Analog (undigested Forced-degradation
compound) solution Human PTH (1-34) -- 1.00 Analog (1) 0.43 0.42
Analog (2) 0.44 0.43 Analog (3) 0.46 0.46 Analog (4) 0.49 0.49
Analog (5) 0.51 0.51 Analog (6) 0.55 0.55 Analog (7) 0.62 0.62
Analog (8) 0.65 0.65 Analog (9) 0.70 0.70
[0317] (2) Structural Estimation Analysis of Each Analog
[0318] Each of the above analogs (undigested compounds) and human
PTH (1-34) standard were digested by trypsin to produce analogs
(digested compounds) and standard solution (digested compound). Ten
of these samples were analyzed by LC/MS/MS under the following
conditions.
[0319] <LC/MS/MS Conditions>
[0320] Detector: Ultraviolet absorptiometer (measurement
wavelength: 210 nm)
[0321] Column: Stainless steel tube 150 mm long having an inner
diameter of 1.5 mm packed with 5 .mu.m of octadecylsilylated silica
gel
[0322] Column temperature: Constant temperature near 40.degree.
C.
[0323] Mobile phase: Mobile phase A: Mixed aqueous solution
containing trifluoroacetic acid (1:1000)
[0324] Mobile phase B: Acetonitrile
[0325] Mobile phase feed: Concentration gradient control is
provided by varying the mixture ratio of mobile phase A and mobile
phase B as follows.
[0326] Time after injection:
TABLE-US-00011 TABLE 9 Concentration gradient control Time after
sample injection (min) Mobile phase A (vol %) Mobile phase B (vol
%) 0-30 95.fwdarw.55 5.fwdarw.45 30-40 55 45
[0327] Flow rate: 0.1 mL/min
[0328] Sample temperature: Constant temperature near 5.degree.
C.
[0329] Detection time: 45 minutes after injection of the sample
solution. However, this is from the back of the solvent peak
[0330] Ionization Mode: ES+
[0331] Each analog (undigested compound) was analyzed by LC/MS
under the following conditions.
[0332] <LC/MS Test Conditions>
[0333] Conditions other than the following were the same as the
LC/MS/MS conditions.
[0334] Mobile phase feed: Concentration gradient control is
provided by varying the mixture ratio of mobile phase A and mobile
phase B as follows.
[0335] Time after injection:
TABLE-US-00012 TABLE 10 Concentration gradient control Time after
sample injection (min) Mobile phase A (vol %) Mobile phase B (vol
%) 0-30 85.fwdarw.55 15.fwdarw.45 30-40 55 45
[0336] <Results>
[0337] The results of structural analysis of the nine analogs in
Test Example 2 were as follows.
[0338] <Human PTH (1-34)>
[0339] Table 11 shows the expected fragments of human PTH (1-34)
produced by trypsin digestion.
TABLE-US-00013 TABLE 11 Expected fragments of human PTH (1-34)
Fragment Estimated No. structure Amino acid sequence T1 PTH (1-13)
Ser-Val-Ser-Glu-Ile-Gln-Leu- Met-His-Asn-Leu-Gly-Lys SEQ ID NO: 1
T2 PTH (14-20) His-Leu-Asn-Ser-Met-Glu-Arg SEQ ID NO: 2 T3 PTH
(21-25) Val-Glu-Trp-Leu-Arg SEQ ID NO: 3 T4 PTH (26) Lys T5 PTH
(27) Lys T6 PTH (28-34) Leu-Gln-Asp-Val-His-Asn-Phe SEQ ID NO:
5
[0340] Table 12 shows the results of measurement of the mass of
each fragment confirmed in LC/MS/MS of the standard solution
(digested compound). The measured values of each fragment in the
standard solution (digested compound) were compared with the
calculated mass, and it was confirmed that five fragments of
estimated structure were obtained in human PTH (1-34).
TABLE-US-00014 TABLE 12 Results of mass measurement of standard
solution (digested compound) Retention Measured mass Calculated
mass Estimated Fragment time Mass (mono.) Mass (mono.) structure
No. 13.423 885.4984 885.4127 PTH (14-20) T2 18.371 871.5030
871.4188 PTH (28-34) T6 18.371 999.6100 999.5138 PTH (27-34) T5-6
21.222 701.4508 701.3861 PTH (21-25) T3 22.618 1454.8715 1454.7551
PTH (1-13) T1
[0341] <Analog 1>
[0342] The analog showing a retention time=0.43 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 1,
and Table 13 shows the results of mass measurement in LC/MS/MS of
analog 1 (digested compound). Changes in mass of +16 Da in T2, +4
Da in T3, and +16 Da in T1 were confirmed in analog 1 (digested
compound) by comparison with the measured values of the relevant
fragments in the standard solution (digested compound).
TABLE-US-00015 TABLE 13 Results of mass measurement of analog 1
(digested compound) Retention Difference time Analog 1 Standard
solution in mass (min) Mass (mono.) Frag. No. Mass (mono.) (F = 0,
4/5) 9.578 901.4622 T2 885.4984 +16 Da 18.409 871.4731 T6 871.5030
0 18.409 999.5720 T5-6 999.6100 0 19.096 705.4228 T3 701.4508 +4 Da
19.323 1470.8297 T1 1454.8715 +16 Da
[0343] Table 14 shows the results obtained by MS/MS analysis of
fragments confirmed to have changes in mass. As a result of
comparison with the standard solution (digested compound), changes
in mass of +16 Da in Met 18 in T2, +4 Da in Trp 23 in T3, and +16
Da in Met 8 in T1 were confirmed.
TABLE-US-00016 TABLE 14 Results of MS/MS analysis of analog 1
(digested compound) Mass observed on MS/MS spectrum Frag- Analog
Standard Amino acid ment 1 solution Estimated with change No.
(mono.) (mono.) structure in mass T2 304.1896 304.1960 PTH (19-20)
Met 18 +16 Da 434.2278 -- PTH (18-20) +16 Da -- 418.2120 PTH
(18-20) T3 271.2070 271.2085 PTH (24-25) Trp 23 +4 Da 478.2979 --
PTH (23-25) +4 Da -- 474.3203 PTH (23-25) T1 568.3508 568.3636 PTH
(9-13) Met 8 +16 Da 715.3823 -- PTH (8-13) +16 Da -- 699.4180 PTH
(8-13)
[0344] Table 15 shows the results obtained by comparing the mass of
analog 1 (undigested compound) obtained by LC/MS with the
calculated value of 4115.1309 of human PTH (1-34). In analog 1
(undigested compound) peaks of +64 Da and +36 Da were confirmed in
comparison to the calculated mass, and the +64 Da peak appeared to
be the main peak based on the size of the peaks, as shown in FIG.
7. The molecular weight of the undigested compound is approximately
4000 Da, but since the mass of a multivalent ion is obtained as the
measured value in LC/MS, an error of about .+-.1 Da was anticipated
in the process of calculating the mass of the undigested compound
from the mass of the multivalent ion. Corrected values are listed
in parentheses for estimated differences in mass when an error
arose in structural analysis. The same is true in subsequent
structural analyses.
TABLE-US-00017 TABLE 15 Results of mass measurement of analog 1
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 19.749 4179.3280 4115.1309 +64 Da 20.130 4151.3674 4115.1309
+36 Da
[0345] Based on the results of MS/MS analysis, it was estimated
that human PTH (1-34)+36 Da=(Met 18+16 Da)+(Trp 23+4 Da)+(Met 8+16
Da). The structure of the +4 Da changed form in Trp is b) in FIG.
6, and the change in mass of its precursor a) was expected to be
+32 Da. Trp 23 was assumed to have changed from a) to b) in the
course of trypsin digestion and other such procedures, and the main
peak of analog 1 (undigested compound) was estimated to be human
PTH (1-34)+64 Da=(Met 18+16 Da)+(Trp 23+32 Da)+(Met 8+16 Da). In
other words, analog 1 was estimated to be human PTH (1-34)-Met 8
[O]-Met 18 [O]-Trp 23 [dioxidation].
[0346] <Analog 2>
[0347] The analog showing a retention time=0.44 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 2,
and Table 16 shows the results of mass measurement in LC/MS/MS of
analog 2 (digested compound). Changes in mass of +16 Da in T2, +4
Da in T3, and +16 Da in T1 were confirmed in analog 2 (digested
compound) by comparison with the measured values of the relevant
fragments in the standard solution (digested compound).
TABLE-US-00018 TABLE 16 Results of mass measurement of analog 2
(digested compound) Retention Difference time Analog 2 Standard
solution in mass (min) Mass (mono.) Frag. No. Mass (mono.) (F = 0,
4/5) 9.58 901.4695 T2 885.4984 +16 Da 18.348 871.4788 T6 871.5030 0
18.348 999.5874 T5-6 999.6100 0 19.059 705.4333 T3 701.4508 +4 Da
19.222 1470.8492 T1 1454.8715 +16 Da
[0348] Changes in mass of +16 Da in Met 18 in T2, +4 Da in Trp 23
in T3, and +16 Da in Met 8 in T1 were confirmed, in the same way as
in analog 1, as a result of MS/MS analysis of fragments confirmed
to have changes in mass. Table 17 shows the results of comparing
the mass of analog 2 (undigested compound) obtained by LC/MS with
the calculated mass of 4115.1309 of human PTH (1-34). In analog 2
(undigested compound), peaks of +24 Da and +92 Da were confirmed in
comparison to the calculated masses, but the reliability of these
confirmed masses appeared to be low since basically no peak shape
was formed, as shown in FIG. 8, and no masses supporting the
results of MS/MS analysis could be obtained.
TABLE-US-00019 TABLE 17 Results of mass measurement of analog 2
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 20.092 4139.2866 4115.1309 +24 Da 20.632 4207.3668 4115.1309
+92 Da
[0349] Based on the results of MS/MS analysis, analog 2 was
estimated to have undergone at least changes in mass of human PTH
(1-34)+36 Da=(Met 18+16 Da)+(Trp 23+4 Da)+(Met 8+16 Da). In other
words, analog 2 was estimated to be human PTH (1-34)-Met 8 [O]-Met
18 [O]-Trp 23 [dioxidation-formic acid elimination].
[0350] <Analogs 3 and 4>
[0351] The peak showing a retention time=0.46 in the column
"Analogs (undigested compounds)" in Table 8 was derived from a
mixture of analogs 3 and 4, as explained below. Table 18 shows the
results of mass measurement in LC/MS/MS of a mixture of analogs 3
and 4 (digested compound). Changes in mass of +16 Da in T2, +4 Da
in T3, and +16 Da in T1 were confirmed in the mixture of analogs 3
and 4 (digested compound) by comparison with the measured values of
the relevant fragments in the standard solution (digested
compound). T2 and T3 fragments not associated with changes in mass
were also confirmed.
TABLE-US-00020 TABLE 18 Results of mass measurement of a mixture of
analogs 3 and 4 (digested compound) Retention Difference time
Analog 3 Standard solution in mass (min) Mass (mono.) Frag. No.
Mass (mono.) (F = 0, 4/5) 9.813 901.4778 T2 885.4984 +16 Da 13.475
885.4846 T2 885.4984 0 18.326 871.4844 T6 871.5030 0 18.326
999.5800 T5-6 999.6100 0 18.959 705.4266 T3 701.4508 +4 Da 19.223
1470.8373 T1 1454.8715 +16 Da 21.195 701.4322 T3 701.4508 0
[0352] Changes in mass of +16 Da in Met 18 in T2, +4 Da in Trp 23
in T3, and +16 Da in Met 8 in T1 were confirmed, in the same way as
in analog 1, as a result of MS/MS analysis of fragments confirmed
to have changes in mass. Table 19 shows the results of comparing
the mass of a mixture of analogs 3 and 4 (undigested compound)
obtained by LC/MS with the calculated mass of 4115.1309 of human
PTH (1-34). In the mixture of analogs 3 and 4 (undigested
compound), peaks of +32 Da, +48 Da, and +20 Da were confirmed in
comparison to the calculated masses, and +32 Da and +48 Da in an
approximate ratio of 1:1 appeared to be the main peak based on the
size of the peaks, as shown in FIG. 9.
TABLE-US-00021 TABLE 19 Results of mass measurement of a mixture of
analogs 3 and 4 (undigested compound) Retention Difference time
Measured mass Calculated mass in mass (min) Mass (mono.) Mass
(mono.) (F = 0, 4/5) 21.062 4147.3692 4115.1309 +32 Da 21.539
4163.3747 4115.1309 +48 Da 21.843 4135.3897 4115.1309 +20 Da
[0353] On the LC/MS/MS chromatogram of a mixture of analogs 3 and 4
(digested compound), T2+16 Da: T2 and T3+4 Da: T3 were each present
in an approximately 1:1 ratio, and T2+16 Da: T3+4 Da: T1+16 Da were
present in an approximately 1:1:2 ratio. Based on the results of
MS/MS analysis, it was estimated that human PTH (1-34)+32 Da=(Met
18+16 Da)+(Met 8+16 Da) and human PTH (1-34)+20 Da=(Trp 23+4
Da)+(Met 8+16 Da). With regard to the latter, Trp 23 was assumed to
have undergone a change of a) to b) in the course of trypsin
digestion and other such procedures, in the same way as analog 1,
and it was estimated that human PTH (1-34)+48 Da=(Trp 23+32
Da)+(Met 8+16 Da). Analogs 3 and 4 were estimated to be human PTH
(1-34)+32 Da and human PTH (1-34)+48 Da, respectively. In other
words, the peak having a relative retention time=0.46 in Table 8
was estimated to be a peak of a mixture containing human PTH
(1-34)-Met 8 [O]-Met 18 [O] and human PTH (1-34)-Met 8 [O]-Trp 23
[dioxidation].
[0354] <Analog 5>
[0355] The analog showing a retention time=0.49 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 5,
and Table 20 shows the results of mass measurement in LC/MS/MS of
analog 5 (digested compound). Changes in mass of +16 Da in T2 and
+4 Da in T3 were confirmed in analog 5 (digested compound) by
comparison with the measured values of the relevant fragments in
the standard solution (digested compound).
TABLE-US-00022 TABLE 20 Results of mass measurement of analog 5
(digested compound) Retention Analog 4 Difference time [sic]
Standard solution in mass (min) Mass (mono.) Frag. No. Mass (mono.)
(F = 0, 4/5) 9.587 901.4664 T2 885.4984 +16 Da 18.321 871.4904 T6
871.5030 0 18.321 999.5957 T5-6 999.6100 0 19.012 705.4388 T3
701.4508 +4 Da 22.561 1454.8492 T1 1454.8715 0
[0356] Changes in mass of +16 Da in Met 18 in T2 and +4 Da in Trp
23 in T3 were confirmed, in the same way as in analog 1, as a
result of MS/MS analysis of fragments confirmed to have changes in
mass. Table 21 shows the results of comparing the mass of analog 5
(undigested compound) obtained by LC/MS with the calculated mass of
4115.1309 of human PTH (1-34). In analog 5 (undigested compound),
peaks of +48 Da and +20 Da were confirmed in comparison to the
calculated masses, and +48 Da appeared to be the main peak based on
the size of the peaks, as shown in FIG. 10.
TABLE-US-00023 TABLE 21 Results of mass measurement of analog 5
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 22.081 4163.3763 4115.1309 +48 Da 22.514 4135.3902 4115.1309
+20 Da
[0357] Based on the results of MS/MS analysis, it was estimated
that human PTH (1-34)+20 Da=(Met 18+16 Da)+(Trp 23+4 Da). Trp 23
was assumed to have undergone a change of a) to b) in the course of
trypsin digestion and other such procedures, in the same way as
analog 1. The main peak of analog 5 (undigested compound) was
estimated to be human PTH (1-34)+48 Da=(Met 18+16 Da)+(Trp 23+32
Da). In other words, analog 5 was estimated to be human PTH
(1-34)-Met 18 [O]-Trp 23 [dioxidation].
[0358] <Analog 6>
[0359] The analog showing a retention time=0.51 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 6,
and Table 22 shows the results of mass measurement in LC/MS/MS of
analog 6 (digested compound). Changes in mass of +16 Da in T2 and
+4 Da in T3 were confirmed in analog 6 (digested compound) by
comparison with the measured values of the relevant fragments in
the standard solution (digested compound). T1 fragments in which
there was no change in mass were also confirmed.
TABLE-US-00024 TABLE 22 Results of mass measurement of analog 6
(digested compound) Retention Analog 5 Difference time [sic]
Standard solution in mass (min) Mass (mono.) Frag. No. Mass (mono.)
(F = 0, 4/5) 9.589 901.4699 T2 885.4984 +16 Da 18.332 871.4912 T6
871.5030 0 18.332 999.5869 T5-6 999.6100 0 19.032 705.4321 T3
701.4508 +4 Da 22.583 1454.8536 T1 1454.8715 0
[0360] Changes in mass of +16 Da in Met 18 in T2 and +4 Da in Trp
23 in T3 were confirmed, in the same way as in analog 1, as a
result of MS/MS analysis of fragments confirmed to have changes in
mass. Table 23 shows the results of comparing the mass of analog 6
(undigested compound) obtained by LC/MS with the calculated mass of
4115.1309 of human PTH (1-34). In analog 6 (undigested compound), a
peak of +20 Da was confirmed in comparison with the calculated
mass.
TABLE-US-00025 TABLE 23 Results of mass measurement of analog 6
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 22.580 4135.6585 4115.1309 +21 Da (+20 Da)
[0361] Based on the results of MS/MS analysis, analog 6 was
estimated to be human PTH (1-34)+20 Da=(Met 18+16 Da)+(Trp 23+4
Da). In other words, analog 6 was estimated to be human PTH
(1-34)-Met 18 [O]-Trp 23 [dioxidation-formic acid elimination].
[0362] <Analog 7>
[0363] The analog showing a retention time=0.55 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 7,
and Table 24 shows the results of mass measurement in LC/MS/MS of
analog 7 (digested compound). A change in mass of +16 Da in T1 was
confirmed in analog 7 (digested compound) by comparison with the
measured values of the relevant fragments in the standard solution
(digested compound).
TABLE-US-00026 TABLE 24 Results of mass measurement of analog 7
(digested compound) Retention Analog 6 Difference time [sic]
Standard solution in mass (min) Mass (mono.) Frag. No. Mass (mono.)
(F = 0, 4/5) 13.405 855.4859 T2 885.4984 0 18.374 871.4885 T6
871.5030 0 18.374 999.5920 T5-6 999.6100 0 19.296 1470.8591 T1
1454.8715 +16 Da 21.225 701.4354 T3 701.4508 0
[0364] A change in mass of +16 Da in Met 8 in T1 was confirmed, in
the same way as in analog 1, as a result of MS/MS analysis of
fragments confirmed to have changes in mass. Table 25 shows the
results of comparing the mass of analog 7 (undigested compound)
obtained by LC/MS with the calculated mass of 4115.1309 of human
PTH (1-34). In analog 7 (undigested compound), a peak of +16 Da was
confirmed in comparison with the calculated mass.
TABLE-US-00027 TABLE 25 Results of mass measurement of analog 7
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 22.353 4131.7101 4115.1309 +17 Da (+16 Da)
[0365] Based on the results of MS/MS analysis, analog 7 was
estimated to be human PTH (1-34)+16 Da=(Met 8+16 Da). In other
words, analog 7 was estimated to be human PTH (1-34)-Met 8 [O].
[0366] <Analog 8>
[0367] The analog showing a retention time=0.62 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 8,
and Table 26 shows the results of mass measurement in LC/MS/MS of
analog 8 (digested compound). A change in mass of +16 Da in T2 was
confirmed in analog 8 (digested compound) by comparison with the
measured values of the relevant fragments in the standard solution
(digested compound).
TABLE-US-00028 TABLE 26 Results of mass measurement of analog 8
(digested compound) Retention Analog 7 Difference time [sic]
Standard solution in mass (min) Mass (mono.) Frag. No. Mass (mono.)
(F = 0, 4/5) 9.709 901.4782 T2 885.4984 +16 Da 18.356 871.4903 T6
871.5030 0 18.356 999.5916 T5-6 999.6100 0 21.228 701.4383 T3
701.4508 0 22.636 1454.8592 T1 1454.8715 0
[0368] A change in mass of +16 Da in Met 18 in T2 was confirmed, in
the same way as in analog 1, as a result of MS/MS analysis of
fragments confirmed to have changes in mass. Table 27 shows the
results of comparing the mass of analog 8 (undigested compound)
obtained by LC/MS with the calculated mass of 4115.1309 of human
PTH (1-34). In analog 8 (undigested compound), a peak of +16 Da was
confirmed in comparison with the calculated mass, as shown in FIG.
13.
TABLE-US-00029 TABLE 27 Results of mass measurement of analog 8
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 23.129 4131.7286 4115.1309 +17 Da (+16 Da)
[0369] Based on the results of MS/MS analysis, analog 8 was
estimated to be human PTH (1-34)+16 Da=(Met 18+16 Da). In other
words, analog 8 was estimated to be human PTH (1-34)-Met 18
[O].
[0370] <Analog 9>
[0371] The analog showing a retention time=0.65 in the column
"Analogs (undigested compounds)" in Table 8 was taken as analog 9,
and Table 28 shows the results of mass measurement in LC/MS/MS of
analog 9 (digested compound). A change in mass of +4 Da in T3 was
confirmed in analog 9 (digested compound) by comparison with the
measured values of the relevant fragments in the standard solution
(digested compound).
TABLE-US-00030 TABLE 28 Results of mass measurement of analog 9
(digested compound) Retention Analog 8 Difference time [sic]
Standard solution in mass (min) Mass (mono.) Frag. No. Mass (mono.)
(F = 0, 4/5) 13.392 885.4876 T2 885.4984 0 18.329 871.4896 T6
871.5030 0 18.329 999.5977 T5-6 999.6100 0 19.023 705.4405 T3
701.4508 +4 Da 22.567 1454.8638 T1 1454.8715 0
[0372] A change in mass of +4 Da in Trp 23 in T3 was confirmed, in
the same way as in analog 1, as a result of MS/MS analysis of
fragments confirmed to have changes in mass. Table 29 shows the
results of comparing the mass of analog 9 (undigested compound)
obtained by LC/MS with the calculated mass of 4115.1309 of human
PTH (1-34). In analog 9 (undigested compound), peaks of +32 Da and
+4 Da were confirmed in comparison with the calculated mass, and
+32 Da appeared to be the main peak based on the size of the peaks,
as shown in FIG. 14.
TABLE-US-00031 TABLE 29 Results of mass measurement of analog 9
(undigested compound) Retention Difference time Measured mass
Calculated mass in mass (min) Mass (mono.) Mass (mono.) (F = 0,
4/5) 23.729 4147.8250 4115.1309 +33 Da (+32 Da) 24.087 4119.8303
4115.1309 +5 Da (+4 Da)
[0373] Based on the results of MS/MS analysis, it was estimated
that human PTH (1-34)+4 Da=(Trp 23+4 Da). Trp 23 was assumed to
have undergone a change of a) to b) in the course of trypsin
digestion and other such procedures, in the same way as analog 1.
The main peak of analog 9 (undigested compound) was estimated to be
human PTH (1-34)+32 Da=(Trp 23+32 Da). In other words, analog 9 was
estimated to be human PTH (1-34)-Trp 23 [dioxidation].
[0374] <Analogs 10 and 11>
[0375] The peak showing a retention time=0.70 in the column
"Analogs (undigested compounds)" in Table 8 was derived from a
mixture of analogs 10 and 11, as explained below. Table 30 shows
the results of mass measurement in LC/MS/MS of a mixture of analogs
10 and 11 (digested compound). Changes in mass of +16 Da and +4 Da
in T3 were confirmed as separate fragments in the mixture of
analogs 10 and 11 (digested compound) by comparison with the
measured values of the relevant fragments in the standard solution
(digested compound).
TABLE-US-00032 TABLE 30 Results of mass measurement of a mixture of
analogs 10 and 11 (digested compound) Retention Analog 9 Difference
time [sic] Standard solution in mass (min) Mass (mono.) Frag. No.
Mass (mono.) (F = 0, 4/5) 13.449 885.4893 T2 885.4984 0 18.337
871.4925 T6 871.5030 0 18.337 999.5985 T5-6 999.6100 0 18.821
717.4391 T3 701.4508 +16 Da 19.032 705.4407 T3 701.4508 +4 Da
22.563 1454.8634 T1 1454.8715 0
[0376] Table 31 shows the results obtained by MS/MS analysis of
fragments confirmed to have changes in mass. A change in mass of
+16 Da in Trp 23 in one T3 was confirmed as a result of comparison
with the standard solution (digested compound). The structure of
the changed form of +16 Da in Trp was expected to be c) in FIG. 6.
Although data that made it possible to specify the amino acids
changed in the other T3 could not be obtained, a change in mass of
+4 Da in Trp 23 was estimated from the results of analysis of
analogs 1-8.
TABLE-US-00033 TABLE 31 Results of MS/MS analysis of a mixture of
analogs 10 and 11 (digested compound) Mass observed on MS/MS
spectrum Frag- Analog 9 Standard Amino acid ment [sic] solution
Estimated with change No. (mono.) (mono.) structure in mass T3
271.2131 271.2085 PTH (24-25) Trp 23 +16 Da 490.3079 -- PTH (23-25)
+16 Da -- 474.3203 PTH (23-25)
[0377] Table 32 shows the results of comparing the mass of a
mixture of analogs 10 and 11 (undigested compound) obtained by
LC/MS with the calculated mass of 4115.1309 of human PTH (1-34). In
the mixture of analogs 10 and 11 (undigested compound), peaks of
+16 Da and +4 Da were observed comparison to the calculated masses,
as shown in FIG. 15.
TABLE-US-00034 TABLE 32 Results of mass measurement of a mixture of
analogs 10 and 11 (undigested compound) Retention Difference time
Measured mass Calculated mass in mass (min) Mass (mono.) Mass
(mono.) (F = 0, 4/5) 22.956 4131.7912 4115.1309 +17 Da (+16 Da)
24.151 4119.8118 4115.1309 +5 Da (+4 Da)
[0378] Based on the results of MS/MS analysis, these were
attributed as human PTH (1-34)+16 Da=(Trp 23+16 Da) and human PTH
(1-34)+4 Da=(Trp 23+4 Da), and the peak showing a retention
time=0.70 in the column "Analogs (undigested compounds)" in Table 8
was estimated to be a mixture of analogs 10 and 11. In other words,
analog 10 was estimated to be human PTH (1-34)-Trp 23
[monoxidation], and analog 11 was estimated to be human PTH
(1-34)-Trp 23 [dioxidation-formic acid elimination].
[0379] <Summary of Structural Analysis>
[0380] Table 33 shows the relative retention time and estimated
structure results of each analog. FIG. 5 shows the oxidation of the
methionine residues in the table, and FIG. 6 shows a), b), and c)
in the table. The relative retention time of each analog in the
table shows the relative retention time taking the retention time
of human PTH (1-34) as 1.
TABLE-US-00035 TABLE 33 Relative retention time and estimated
structure of each analog Relative Amino Summary of change retention
acid Change Nature of No. time changed in mass change Name of
analog (1) 0.42 Met 8 16 Da Oxidation Human PTH (1-34)-Met 8 [O]-
Met 18 16 Da Oxidation Met 18 [O]-Trp 23 Trp 23 32 Da a)
[dioxidation] (2) 0.43 Met 8 16 Da Oxidation Human PTH (1-34)-Met 8
[O]- Met 18 16 Da Oxidation Met 18 [O]-Trp 23 Trp 23 4 Da b)
[dioxidation-formic acid elimination] (3) 0.46 Met 8 16 Da
Oxidation Mixture containing human Met 18 16 Da Oxidation PTH
(1-34)-Met 8 [O]-Met 18 0.46 Met 8 16 Da Oxidation [O] and human
PTH (1-34)- Trp 23 32 Da a) Met 8 [O]-Trp 23 [dioxidation] (4) 0.49
Met 18 16 Da Oxidation Human PTH (1-34)-Met 18 Trp 23 32 Da a)
[O]-Trp 23 [dioxidation] (5) 0.51 Met 18 16 Da Oxidation Human PTH
(1-34)-Met 18 Trp 23 4 Da b) [O]-Trp 23 [dioxidation- formic acid
elimination] (6) 0.55 Met 8 16 Da Oxidation Human PTH (1-34)-Met 8
[O] (7) 0.62 Met 18 16 Da Oxidation Human PTH (1-34)-Met 18 [O] (8)
0.65 Trp 23 32 Da a) Human PTH (1-34)-Trp 23 [dioxidation] (9) 0.7
Trp 23 16 Da c) Mixture containing human 0.7 Trp 23 4 Da b) PTH
(1-34)-Trp 23 [monoxidation] and human PTH (1-34)-Trp 23
[dioxidation-formic acid elimination]
INDUSTRIAL APPLICABILITY
[0381] Since a freeze-dried preparation containing high-purity PTH
peptide is provided by the present invention, the present invention
can be used in the pharmaceutical manufacturing industry.
EXPLANATION OF SYMBOLS
[0382] 1: Large door [0383] 2: Small door [0384] 3: Sub-door (open)
[0385] 4: Sub-door (closed) [0386] 5: Airflow-adjusting cover
Sequence CWU 1
1
5113PRTArtificial SequenceTrypsin-digested fragment of hPTH(1-34)
1Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys 1 5 10
27PRTArtificial SequenceTrypsin-digested fragment of hPTH(1-34)
2His Leu Asn Ser Met Glu Arg 1 5 35PRTArtificial
SequenceTrypsin-digested fragment of hPTH(1-34) 3Val Glu Trp Leu
Arg 1 5 434PRTArtificial SequencehPTH(1-34) 4Ser Val Ser Glu Ile
Gln Leu Met His Asn Leu Gly Lys His Leu Asn 1 5 10 15 Ser Met Glu
Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val His 20 25 30 Asn
Phe 57PRTArtificial SequenceTrypsin-digested fragment of hPTH(1-34)
5Leu Gln Asp Val His Asn Phe 1 5
* * * * *