PROCESS AND CONTAINER FOR FREEZE DRYING UNDER STERILE CONDITIONS

PROCESSUS ET CONTENANT UTILISES POUR LA LYOPHILISATION DANS DES CONDITIONS STERILES

English Abstract

The present invention provides a process for
freeze drying of especially biologically or pharma-
ceutical material under sterile conditions, wherein
the material to be dried is introduced into a con-
tainer the sides of which consist at least partly of
a hydrophobic, porous, micro-organism-impermeable,
water vapour-permeable membrane, the container is
tightly closed and the material is subsequently freeze
dried in the closed container under the usual
conditions.
The present invention also provides a container
for freeze drying materials under sterile conditions,
wherein the sides of the container consist at least
partly of a hydrophobic, porous, germ-impermeable,
water vapour-permeable membrane.

Claims

12

The embodiments of the invention in which an exclusive
property or privilege is claimed are defined as
follows:

1. A process for freeze drying of material
under sterile conditions, wherein the material to be
dried is introduced into a container the container
having sides which consist at least partly of two
walls connected hermetically and pressure-proof with
one another on their edges, one wall of which is made
from liquid-tight material and the other wall of a
hydrophobic, porous, germ-tight water vapour-permeable
material, the container is tightly closed and the
material is subsequently freeze dried in the closed
container.

2. A process according to claim 1, wherein said
membrane has pores with a size of < 0.5 µm.

3. A process according to claim 2, wherein said
membrane has pores with a size of < 0.2 µm.

4. A process according to claim 1, 2 or 3,
wherein said membrane is a film having properties
according to German Industrial Standard DIN 58 953.

5. A process according to claim 1, 2 or 3,
wherein said membrane is of semi-permeable paper.

6. A process according to claim 5, wherein the
semi-permeable paper is made of cellulose or a cellu-
lose derivative.

7. A process according to claim 6, wherein said
membrane is of cellulose acetate.

8. A process according to claim 4, wherein said
membrane is of semi-permeable paper.


13

9. A process according to claim 8, wherein the
semi-permeable paper is made of cellulose or a cellu-
lose derivative.

10. A process according to claim 9, wherein said
membrane is of cellulose acetate.

11. A process according to claim 1, 2 or 3,
wherein the membrane used is a film of a polymer com-
pound.

12. A process according to claim 8, wherein the
polymer compound is polytetrafluoroethylene or
polypropylene.

13. A process according to claim 4, wherein the
membrane used is a film of a polymer compound.

14. A process according to claim 13, wherein the
polymer compound is polytetrafluoroethylene or
polypropylene.

15. A process according to claim 1, 2, 3, 6, 7,
8, 9, 10, 12, 13 or 14, wherein said container is in
the form of a tube or bag.

16. A process according to claim 4, wherein said
container is in the form of a tube or bag.

17. A process according to claim 5, wherein said
container is in the form of a tube or bag.

18. A process according to claim 11, wherein
said container is in the form of a tube or bag.

14

19. A process according to claim 15, wherein the
tube or bag has a water-impermeable wall which is
tightly connected with a further wall formed by the
membrane.

20. A process according to claim 16, 17 or 18,
wherein the tube or bag has a water-impermeable wall
which is tightly connected with a further wall formed
by the membrane.

21. A process according to claim 1, 2, 3, 6, 7,
8, 9, 10, 12, 13 or 14, wherein said container is a
flask, ampoule or vial which is closed with the mem-
brane.

22. A process according to claim 4, wherein said
container is a flask, ampoule or vial which is closed
with the membrane.

23. A process according to claim 1, 2, 3, 6, 7,
8, 9, 10, 12, 13 or 14, wherein the container is a
trough which is tightly connected with the membrane as
covering.

24. A process according to claim 4, wherein the
container is a trough which is tightly connected with
the membrane as covering.

25. A process according to claim 1, 2, 3, 6, 7,
8, 9, 10, 12, 13, 14, 16, 17, 18 or 19, wherein said
material is a biological or pharmaceutical material.

26. A process according to claim 4, wherein said
material is a biological or pharmaceutical material.

15

27. A process according to claim 5, wherein said
material is a biological or pharmaceutical material,

28. A process according to claim 11, wherein
said material is a biological or pharmaceutical mate-
rial.

29. A process according to claim 15, wherein
said material is a biological or pharmaceutical mate-
rial.

30. A process according to claim 20, wherein
said material is a biological or pharmaceutical mate-
rial.

31. A process according to claim 21, wherein
said material is a biological or pharmaceutical mate-
rial.

32. A process according to claim 22, wherein
said material is a biological or pharmaceutical mate-
rial.

33. A process according to claim 23, wherein
said material is a biological or pharmaceutical mate-
rial.

34. A process according to claim 24, wherein
said material is a biological or pharmaceutical mate-
rial.

35. A process according to claim 1, 2, 3, 6, 7,
8, 9, 10, 12, 13, 14, 16, 17, 18, 19, 27, 28, 29, 30,
31, 32, 33 or 34, wherein said container is in a non-
sterile environment and introduction of the material
into the container, closing of the container and

16

freeze drying of the material in the closed container
is conducted in said non-sterile environment.

36. A container for freeze drying materials
under sterile conditions, the container having sides
which consist at least partly of two walls connected
hermetically and pressure-proof with one another on
their edges, one wall of which is made from liquid-
tight material and the other wall of a hydrophobic,
porous, germ-tight water vapour-permeable material.

37. A container according to claim 36, in the
form of a bag.

38. A container according to claim 36, in the
form of a tube.

39. A container according to claim 36, in the
form of a trough which is tightly connected with said
membrane as a covering.

40. A container according to claim 36, in the
form of a flask, ampoule or vial closed with the mem-
brane.

41. A container according to claim 36, 37, 38,
39 or 40, wherein pores of the membrane have a size of
?0.5 µm.

42. A container according to claim 41, wherein
said size is ?0.2 µm.

43. A container according to claim 36, 37, 38,
39, 40 or 42, wherein said membrane is a film with
properties according to DIN 58953.

17

44. A container according to claim 36, 37, 38,
39, 40 or 42, wherein said membrane is of semi-
permeable paper.

45. A container according to claim 44, wherein
said membrane is of cellulose or a cellulose
derivative.

46. A container according to claim 45, wherein
said membrane is of cellulose acetate.

47. A container according to claim 36, 37, 38,
39, 40 or 42, wherein said membrane is a film of a
polymer compound.

48. A container according to claim 47, wherein
said polymer compound is polytetrafluoroethylene or
polypropylene.

18

49. The method of freeze drying biological or
pharmaceutical material under sterile conditions, which
comprises introducing said material into a container the
sides of which consist at least partly of a hydrophobic,
porous, micro-organism-impermeable and water vapour-
permeable membrane, tightly closing the container and
freeze drying said material under sterile conditions
inside the closed container in a non-sterile ambient
environment.

50. The method of claim 49, wherein the membrane
has pores with a size of 0.5 µm.
51. The method of claim 50, wherein the membrane
has pores with a size of 0.2 µm.

52. The method of claim 49, 50 or 51, wherein
said membrane is semi-permeable paper.

53. The method of claim 52, wherein the semi-
permeable paper is made of cellulose or a cellulose
derivative.
54. The method of claim 53, wherein the membrane
is of cellulose acetate.

55. The method of claim 49, wherein the membrane
is a film of a polymer compound.

56. The method of claim 55, wherein the polymer
compound is polytetrafluoroethylene or polypropylene.

57. The method of claim 49, 50, 51, 53, 54, 55 or
56, wherein the container is in the form of a tube or
bag.

58. The method of claim 57, wherein the tube or
bag comprises a water-impermeable wall which is tightly
connected with a further wall formed by the membrane.


19
59. The method of claim 49, 50, 41, 53, 54, 55
or 56, wherein the container is a flask, ampoule or
vial which is closed with the membrane.

60. The method of claim 49, 50, 51, 53, 54, 55
or 56, wherein the container is a trough which is
tightly connected with the membrane as covering.

61. A process according to claim 5, wherein said
container is a flask, ampoule or vial which is closed
with the membrane.

62. A process according to claim 11, wherein
said container is a flask, ampoule or vial which is
closed with the membrane.

63. A process according to claim 5, wherein the
container is a trough which is tightly connected with
the membrane as covering.

64. A process according to claim 11, wherein the
container is a trough which is tightly connected with
the membrane as covering.

Description

1337974
--2--

The present invention is concerned with a process
for freeze drying under sterile conditions, as well as
with a container for the carrying out of the process.
The present invention is especially concerned with the
drying of biological and/or pharmaceutical material-

In the case of biological and pharmaceutical.naterials, it is frequently ~ecessary to store the
rnaterials completely dry until they are used. Thes~
sensitive substances are mostly only obtainable by
freeze drying. Furthermore, as a rule, it is necessary
to keep these substances completely free from micro-
organisms not only because OL the deco,nposition of
biological substances brought about by micro-organisms
but also in order to prevent possible infections in the
case of their use.
The freeze drying of biological and pharmaceutical
-.naterials is generally known (see also Ullmanns
Enzyklopadie der Technischen Che-,nie, 3rd edition,
Vol. I, p. 556 et seq.). In order to avoid a contamin-

ation of the dried material with micro-organisms and
other contaminants, laborious apparatus and process-
technical measures have to be made.
In the case of drying phari~aceutical preparations
in ampoules or small bottles, the procedure is, for
example, to provide small bottles which contain the
frozen material with a bacterial filter and to dry the
material in the small bottles in a first drying step to


1337974
--3--


such an extent that the sublimation of the frozen
solvent is concluded.
Subsequently, in a second drying stage, i.e. the
so-called ?ost or residual drying, the still remaining
residual moisture is removed from the material. Since
this second drying step is usually carried out in a
special apparatus, the ampoules or vials must be
removed from the first drying apparatus in a further
working step which is prone to contamination and
introduced into the second drying apparatus. For this
purpose, the bacterial filter is removed and re?laced
by an aluminium cap provided with a rubber diaphragm
and a hollow needle. After a residual drying for
several days depending upon the nature of the -,naterial
to be dried, the drying chamber is filled with an inert
gas and with slight overpressure and the diaphrag
opening closed as vapour-tightly as ?ossible by a
grouting mass.
Since the speed of sublimation in the case of
this type of freeze drying is only about half as great
as that of openly spread out material, the freeze
drying of biological and pharmaceutical material is
also carried out on plates under sterile conditions.
A solution of the ~aterial to be dried is thereby first
sterilised, for e~alnple by filtration over a sterile
filter, subsequently poured under sterile conditions on
to plates and fr~eze dried by means of known methods.


1~3797~
--4--

However, a prerequisite of this process is that the
whole of the freeze drying plant can be sterilised.
~urthermore, it is also necessary to keep the surround-
ings of the drying plant free from micro-organisms.
After drying has taken place, it is necessary to
remove the material in the drying plant itself or in
its surroundings with mechanical processes from the
plates under sterile conditions and to fill it into
also sterile storage containers. This process requires
laborious plant and sterile charnbers, as well as an
especially careful working with the material to be
dried or already dried until it is confectioned ready
for use.

The present invention seeks to
overcome the above-mentioned disadvantages and to
provide a simple process with the help of which freeze
dried material can be obtained without the above-
mentioned laborious sterility requirements for the
drying plant, as well as for the space surrounding
this.
Thus, according to the present invention, there
is provided a process for freeze drying of especially
biological or pharmaceutical ,naterial under sterile
conditions, wherein the ~aterial to be dried is intro-

duced under sterile conditions into a container, thesides of which consist at least partly of a hydrophobic
germ-impermeable, porous, micro-organisrn-imper~eable



B~

~ 5 ~ 133797~

membrane which is permeable to water in vapour form, the
container is closed micro-organism-impermeably and
tightly, especially cemented or welded, and the material
is subsequently freeze dried directly in the closed
container under the usual conditions, and more
especially the freeze drying of the material is carried
out under sterile conditions inside the closed container
in a non-sterile ambient environment.
The present invention is based on the surprising
finding that, contrary to expectations, the vapour flow
resulting by the sublimation of solvent molecules, which
flows from the material to be dried to a condenser, is
hindered only to a small extent by the membrane used in
the process according to the present invention. Thus,
surprisingly, the freeze drying of material which is
enclosed by the membrane proceeds almost equally quickly
as the freeze drying of the same material when open and
non-packed.
The membranes used according to the present
invention are hydrophobic membranes which contain pores
which, on the one hand, are permeable for water vapour
but, on the other hand, are so small that micro-
organisms can no longer pass through. Such pores
preferably have a size of < 0.5 ~m. and especially of <
0.2 ~m. According to the present invention, membranes
are preferably used which, under the particular process
conditions, are also tearproof even in a wet state.
However, the process according to the present invention
can also be carried out with less stable membranes
provided that these are strengthened with a carrier
C

1337974
--6--

material or are not excessively mechanically stressed.
~ he particularly selected proportion of the
membrane on the wall surface of the container used in
the process according to the present invention depends
upon the particular selected conditions and the drying
period and can easily be ascertained by means of si-nple
experiments. In one e~,nbodiment preferred according to
the present invention, the whole wall surface consists
of the membrane film and in a further preferred embodi-

ment about one half of the wall surface consists of themembrarle film. Surprisingly, the process according to
the present invention can also be advantageously
carried out when the wall surface also only consists
of up to 10% of the me.nbrane film.
In particular, there can be used semi-permeable
papers of cellulose and usual cellulose derivatives,
sucn as cellulose acetate. According to the present
invention, rneinbranes of films of polymer co,npounds,
for example ?olytetrafluoroethylene or polypropylene,
can also be advantageously used. Films of sterilis-
ation paper according to German In dustrial Standard
DI`L~ 5~ 953 are also quite especially useful as water
vapour-per-lleable rnernbranes which standard thus counts
as a part of the present description. In further
preferred embodiments of the present invention,
Gorete,~*and sir,.ilar merllbranes or also co-.nmercially
available film tubes can be used, such as are marketed


Trade Mark

IB

1337979


by the firm Vihuri OY, Wipack, Finland, under the
designation "`~ediplast". In principle, all film
membrane can be used, regardless of their components,
provided that they fulfil the requirements with re~ard
to raicro-organism impermeability, air pen~eability and
especially strength given in German Industrial Standard
DIN 5~ 953-

In a preferred embodiment, the process accordingto the present invention is carried out with the use
of a bag or tube which preferably consists of two walls
hennetically and tightly connected with on~ another on
their edges, one wall of which consists of a liquid-
tight material and the other wall of the me.tlbrane.
The membrane is preferably welded or glued with
the vessel. Accordin~ to the present invention,
trou~hs are especially pre~erred as the vessels.
In a further preferred embodi~nent, the trough
consists of liquid-impermeable synthetic resin and
preferably has a wall thickness of 0.5 to 1 mm.
The most favourable drying conditions, such as
pressure, temperature and amount, depend upon the
particular material to be dried and the thickness of
the .nembrane, as well as upon the size and number of
the pores thereof and must be determined by usual and
simple experimentation for the particular material and
the packing.
The following Examples are given for the purpose
of illustrating the present invention:

* Trade Mark

1337974
--8--

Example 1.
The testing of the micro-organism impermeability
of a membrane was so carried out according to German
Industrial Standard DIN 58 953 that micro-organisms in
water drops were applied to test pieces and, after
drying the water drops, it was ascertained whether
micro-organisms have passed through to the under side
of the test pieces.
The membrane film to be tested was cut up into
lG squares of about 50 rnm. edge length and the test pieces
were sterilised and dried. Each test piece of the
sterilised mernbrane was placed on a sterilised substrate
with the side which can be contaminated in the case of
use upwardly and inoculated with 5 drops each of
0.1 ml. (corresponding to 106 to 107 micro-organisms).
The test pieces were stored at a temperature of 20 to
25C. under a relative atmospheric humidity of 40 to
60%. The drops must be completely dried within 6 hours.
Each test piece was placed with the inoculated surface
upwardly on the surface of a blood agar plate (1.5%
agar) so that the whole film surface came into contact
with the agar. ~fter 5 to 6 seconds, the paper was
removed and the plates were incubated for 16 to 25 hours
at 37C. If the agar plates treated with such film
samples s"how no growth, the film is regarded as being
sufficiently impermeable to micro-organisms. Further
statements regarding the testing of the impermeability


133797~


of membranes to micro-organisms and especially the
preparation of test micro-organism suspensions, can be
taken from part 6 of German Industrial Standard DI~
53 953.
Example 2.
A nutrient solution was produced whicn consisted
of 10 g. peptone, 5 g. glucose, 5 g. sodium chlori~,
Q.OS4 g. monopotassiu~ dihydrogen phosphate, 0.187 g.
disodium hydrogen phosphate dihydrate and pyrogen-free
water ad 1.0 litre and which had been adjusted to pH
7Ø Subsequently, it was end-sterilised in a closed,
piercable bottle.
For the reception of the sterile nutrient
solution to be lyophilised, there was prepared a trans-

parent sterile bag consisting of a transparent filmand an appropriate paper. For this purpose, a piece
with a length of 800 mm. was cut off from a commercially
available roll of transparent sterilisation bag film
of the firm Wipak Medical, type Steri-King*R 47 which
is available in the form of a tube, i.e. is welded on
both sides but is otherwise open, the roll having a
width of 400 mm. This tube was welded on both of the
open sides with a commercially available film-welding
apparatus to form a bag. Subsequently, this bag was
sterilised in an autoclave with filter programme at
123C. and 2 bar vapour pressure, the sterile bag was
placed with the transparent film downwardly for better

i B * Trade Mark

1337~7~
-10-
handling in a non-sterile sheet metal trough (VA sheet
metal, dimensions: length 800 mm., breadth 400 mm.,
height 30 mm.) and opened in a laminar flowbox under
sterile conditions with disinfected scissors by cutting
off of a corner. Through this opening of about 30 mm.
between the film and the paper was introduced 1.5 litres
of sterile nutrient solution via a sterile tube pushed
into the opening. The so filled bag was again closed
in the laminar flowbox under sterile conditions by
means of a commercially available film welding device
by welding over the corner.
The whole assembly (sheet metal trough, bag and
sterile nutrient solution) was applied to a plate
pre-cooled to -45C. of a commercially available, non-

sterilisable freeze drying apparatus of the firmEdwards + Kniese with a total positioning surface of
1.5 m and the solution frozen in. After complete
freezing in of the solution under non-sterile
conditions, it was freeze dried at a pressure of 10 1
mm.Hg and a plate temperature of 22C. and the product
post-dried at 10 3 mm.Hg, again under non-sterile
conditions. The total drying time was about 72 hours.
The so obtained freeze-dried material, present
as a pale brown powder in the transparent sterilisation
bag, was subsequently introduced into a laminar flowbox
and dissolved in 1.5 litres of sterile water. For this
purpose, the intended puncturing point was disinfected


1337974


with alcohol on the paper side, by means of a sterile
canula and appropriate sterile syringe a total of
1.5 litres of sterile water was introduced into the
bag, the dried material dissolved and the solution
transferred into a sterile bottle. This solution was
incubated for 4 days at 37C. and subsequently the
micro-organism count of the incubated solution deter-
mined by the membrane filter method.
It was shown that no micro-organisms had been
entrained by the freeze drying.