U.S. patent application number 11/084223 was filed with the patent office on 2005-07-28 for methods of preventing aggregation of various substances upon rehydration or thawing and compositions obtained thereby.
This patent application is currently assigned to Elan Drug Delivery Limited. Invention is credited to Colaco, Camilo, Kampinga, Jaap, Roser, Bruce J., Smith, Christopher.
Application Number | 20050163750 11/084223 |
Document ID | / |
Family ID | 22958303 |
Filed Date | 2005-07-28 |
United States Patent
Application |
20050163750 |
Kind Code |
A1 |
Roser, Bruce J. ; et
al. |
July 28, 2005 |
Methods of preventing aggregation of various substances upon
rehydration or thawing and compositions obtained thereby
Abstract
The present invention encompasses methods to prevent aggregation
of a wide variety of substances during freezing/thawing and/or
dehydrating/rehydrating. The substances thus obtained and
compositions comprising the substances are also encompassed by the
invention.
Inventors: |
Roser, Bruce J.; (Cambridge,
GB) ; Colaco, Camilo; (Cambridge, GB) ;
Kampinga, Jaap; (Groningen, NL) ; Smith,
Christopher; (Huntingdon, GB) |
Correspondence
Address: |
MORRISON & FOERSTER LLP
755 PAGE MILL RD
PALO ALTO
CA
94304-1018
US
|
Assignee: |
Elan Drug Delivery Limited
Nottingham
GB
|
Family ID: |
22958303 |
Appl. No.: |
11/084223 |
Filed: |
March 18, 2005 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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11084223 |
Mar 18, 2005 |
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09836625 |
Apr 16, 2001 |
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6890512 |
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09836625 |
Apr 16, 2001 |
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08252967 |
Jun 2, 1994 |
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Current U.S.
Class: |
424/85.2 ;
424/85.4; 514/11.4; 514/171; 514/2.4; 514/44R; 514/5.9; 514/53;
514/7.6 |
Current CPC
Class: |
A61K 9/0019 20130101;
Y02A 50/465 20180101; C07K 1/00 20130101; C07K 14/61 20130101; A61K
47/26 20130101; A61K 35/18 20130101; Y02A 50/30 20180101 |
Class at
Publication: |
424/085.2 ;
424/085.4; 514/002; 514/053; 514/171; 514/044; 514/003 |
International
Class: |
A61K 038/20; A61K
038/22; A61K 038/21; A61K 031/7012; A61K 048/00; A61K 038/28; A61K
031/56 |
Claims
We claim:
1. A method of reducing aggregation during dehydration and
rehydration of substances comprising the steps of adding to a
solution or suspension of the substances an amount of trehalose
sufficient to prevent aggregation upon rehydration; and dehydrating
the solution or suspension.
2. The method according to claim 1 wherein the substances are
selected from the group consisting of therapeutic, prophylactic and
diagnostic.
3. The method according to claim 2 wherein the substances are
therapeutic and are biological modifiers.
4. The method according to claim 3 wherein the biological modifier
is selected from the group consisting of proteins and peptides,
steroid hormones, oligosaccharides, nucleic acids and small
molecules.
5. The method according to claim 4 wherein the proteins are
selected from the group consisting of growth hormones, growth
factors, insulin, monoclonal antibodies, interleukins and
interferons.
6. The method according to claim 5 wherein the substance is human
growth hormone.
7. The method according to claim 4 wherein the steroid hormones are
selected from the group consisting of estrogen, progesterone and
testosterone.
8. The method according to claim 2 wherein the substances are
prophylactic substances and are aluminum based adjuvants.
9. The method according to claim 8 further comprising the step of
incorporating the adjuvants into vaccines.
10. The method according to claim 9 wherein the vaccines are
diphtheria/tetanus/pertussis (DTP) or inactivated poliovaccine.
11. The method according to claim 10 wherein the vaccine is
DTP.
12. The method according to claim 2 wherein the substance is
diagnostic and is selected from the group consisting of colloidal
gold, polystyrene latex, fixed erythrocytes and monoclonal
antibodies.
13. The method according to claim 12 wherein the substance is red
blood cells, further comprising the step of fixing the red blood
cells prior to adding trehalose.
14. The method according to claim 13 wherein the fixing is by
glutaraldehyde.
15. The method according to claim 1 wherein the trehalose is added
in an amount to obtain a final concentration of from about 1% to
50% (w/v).
16. The method according to claim 1 wherein the trehalose is added
in an amount to obtain a final concentration of from about 5% to
25% (w/v).
17. The method according to claim 1 wherein the dehydration step
occurs by lyophilization, drying at ambient conditions or drying
under reduced vapor pressure.
18. The method according to claim 17 wherein the drying at reduced
vapor pressure occurs at room temperature or at a temperature
elevated above room temperature but below a temperature at which
degradation or chemical change of the substance occurs.
19. The method according to claim 1 further comprising the step of
rehydrating the substance to obtain a solution or suspension of
substantially nonaggregated substance.
20. A method of reducing aggregation of substances in solution or
suspension during freezing comprising the steps of: adding to the
solution or suspension of the substance an amount of trehalose
sufficient to prevent aggregation during freezing; and freezing the
solution or suspension.
21. The method according to claim 18 wherein the substances are
selected from the group consisting of therapeutic, prophylactic and
diagnostic.
22. The method according to claim 21 wherein the substance is
therapeutic and is a biological response modifier.
23. The method according to claim 22 wherein the biological
modifier is selected from the group consisting of proteins and
peptides, steroid hormones, oligosaccharides, nucleic acids and
small molecules.
24. The method according to claim 23 wherein the proteins are
selected from the group consisting of growth hormones, growth
factors, insulin, monoclonal antibodies, interleukins and
interferons.
25. The method according to claim 24 wherein the substance is human
growth hormone.
26. The method according to claim 23 wherein the steroid hormones
are selected from the group consisting of estrogen, progesterone
and testosterone.
27. The method according to claim 21 wherein the substances are
prophylactic substances and are aluminum based adjuvants.
28. The method according to claim 27 further comprising the step of
incorporating the adjuvants into vaccines.
29. The method according to claim 28 wherein the vaccines are
diphtheria/tetanus/pertussis (DTP) or
diphtheria/tetanus/pertussis/inacti- vated poliovaccine
(DTP/IPV).
30. The method according to claim 29 wherein the vaccine is
DTP/IPV.
31. The method according to claim 20 wherein the substance is
diagnostic and is selected from the group consisting of colloidal
gold, polystyrene latex, fixed erythrocytes and monoclonal
antibodies.
32. The method according to claim 20 wherein the trehalose is added
in an amount to attain of from about 1% to 50% (w/v).
33. The method according to claim 32 wherein the trehalose is added
in an amount to attain of from about 5% to 25% (w/v).
34. The method according to claim 20 further comprising the step of
thawing the solution or suspension to obtain a solution or
suspension of substantially nonaggregated substance.
35. An aqueous composition comprising a substance and an amount of
trehalose sufficient to prevent substantial aggregation of the
substance upon freezing and thawing or dehydrating and
rehydrating.
36. A frozen composition comprising a substance and an amount of
trehalose sufficient to prevent substantial aggregation of the
substance upon thawing.
37. A dehydrated composition comprising a substance and an amount
of trehalose sufficient to prevent aggregation of the substance
upon rehydration.
38. A composition obtained by the method according to claim 1.
39. A composition obtained by the method according to claim 13.
40. A composition obtained by the method according to claim 19.
41. A composition obtained by the method according to claim 20.
42. A composition obtained by the method according to claim 34.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to methods of preventing the
formation of aggregates of various substances upon dehydration and
rehydration and upon freezing and thawing. Compositions obtained
thereby are also encompassed by the invention.
BACKGROUND F THE INVENTION
[0002] Storage and processing of a wide range of substances in a
dehydrated or frozen form is necessary to retain activity, prevent
degradation products from forming and to facilitate handling and
transport. Unfortunately, upon rehydration or thawing, many
substances tend to aggregate, thereby decreasing their effective
concentration and often rendering them useless or forming harmful
byproducts.
[0003] Various methods have been tried to prevent or eliminate such
aggregation. For instance, detergents and chaotropic agents are
often used to prevent aggregation of proteins in solution. These
agents are thought to prevent aggregation mediated by hydrophobic
interactions and thus are limited to prevention of aggregation due
to this cause. See, e.g., Tanford and Reynolds (1976) Biochim.
Biophys. Acta. 457:133; and Tanford, "The Hydrophobic Effect", 2nd
Ed., Wiley, N.Y. (1980). Such agents may also not be suitable for
use where the substances are to be formulated into therapeutic
compositions as they may cause adverse reactions. Aluminum salts in
solution are in the form of a highly hydrated colloidal gel and
carry a surface charge at any pH outside their isoelectric point.
Since each colloidal particle carries the same charge, they
mutually repel each other and thus naturally form a stable
colloidal gel. When the hydration shell is removed (e.g., by
freezing or drying) the particles can contact each other and the
surface energy causes aggregation.
[0004] Trehalose,
.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside, is a naturally
occurring, non-reducing disaccharide which was initially found to
be responsible for protection of intact plant cells from
desiccation. Trehalose has been shown to be useful in preventing
denaturation of proteins viruses and foodstuffs during desiccation.
U.S. Pat. Nos. 4,891,319; 5,149,653; 5,026,566; Blakeley et al.
(1990) Lancet 336:854-855; Roser (1991) Trends in Food Sci. and
Tech. pp.166-169; Colaco et al. (1992) Biotechnol. Internat., pp.
345-350; Roser (1991) BioPharm. 4:47-53; and Colaco et al. (1992)
Bio/Tech. 10:1007-1011.
[0005] In the field of protein purification it would be
particularly useful to eliminate or prevent the tendency of
proteins to aggregate upon rehydration and thawing. This is
especially important in the area of biopharmaceuticals where the
proteins are often used as an ongoing basis of treatment. In the
case where protein aggregates form and are injected into a patient,
antibodies may form to the protein which diminish the effectiveness
of the treatment.
[0006] Thus, it would be useful to prevent aggregation of a wide
variety of substances particularly those useful in medicine.
SUMMARY OF THE INVENTION
[0007] The invention encompasses a method of reducing aggregation
during dehydration and rehydration of substances comprising the
steps of adding to a solution or suspension of the substances an
amount of trehalose sufficient to prevent aggregation upon
rehydration; and dehydrating the solution or suspension. The
invention also encompasses the compositions obtained thereby.
[0008] The invention further encompasses rehydrating the solution
or suspension to obtain a composition substantially lacking
aggregates of the substance. The compositions obtained thereby are
also encompassed by the invention.
[0009] The invention further encompasses a method of reducing
aggregation of substances in solution or suspension during freezing
comprising the steps of adding to the solution or suspension of the
substance an amount of trehalose sufficient to prevent aggregation
during freezing; and freezing the solution or suspension. The
invention also comprises the compositions obtained thereby.
[0010] The invention further comprises the step of thawing the
frozen solution or suspension to obtain a composition substantially
lacking aggregates of the substance. The compositions obtained
thereby are also encompassed by the invention.
[0011] A wide variety of substances are suitable for use in the
invention including, but not limited to, therapeutic, prophylactic
and diagnostic.
[0012] When the substance is red blood-cells, the method may
further comprise the step of fixing the red blood cells prior to
adding trehalose. Fixing of red blood cells can be done by any
known method including, but not limited to, formaldehyde and
glutaraldehyde.
BRIEF DESCRIPTION OF THE DRAWINGS
[0013] FIG. 1 is a bar graph depicting the percent height of
sedimentation of aluminum phosphate per column after 24 hours.
Columns labeled with + and - symbols were dried in the presence and
absence of trehalose respectively. Tv stands for vacuum drying, Tfd
stands for freeze drying, Tfz strands for freezing, T4fz stands for
freeze thawing four times and Tw stands for aqueous samples.
[0014] FIG. 2 is a bar graph depicting the percent height of
sedimentation of aluminum phosphate per column after 5.5 hours.
Prior to testing, samples were stored for one week at 45.degree. C.
The abbreviations are the same as those in FIG. 1.
[0015] FIG. 3 is a bar graph depicting the percent height of
sedimentation of aluminum hydroxide after 24 hours. The numbers
refer to the-series as described in Example 3, d stands for vacuum
drying, w stands for aqueous control, and f stands for
freezing.
DETAILED DESCRIPTION OF THE INVENTION
[0016] The present invention encompasses a method of reducing
aggregation during dehydration and rehydration of substances by
adding to a solution or suspension of the substances an amount of
trehalose sufficient to prevent aggregation upon rehydration; and
dehydrating the solution or suspension.
[0017] The invention further encompasses a method of reducing
aggregation of substances in solution or suspension during freezing
and thawing comprising the steps of adding to the solution or
suspension of the substance an amount of trehalose sufficient to
prevent aggregation during freezing and thawing; and freezing the
solution or suspension.
[0018] The term "aggregation" as used herein refers to the
interaction of two or more molecules of a substance such that they
no longer behave as monomers but as dimers, trimers or other
multimeric forms. Reducing aggregation decreases the concentration
of multimeric forms compared to substances dehydrated and
rehydrated or frozen and thawed in the absence of trehalose. A
substance substantially free of aggregates or substantially
nonaggregated is one which, upon rehydration or thawing, contains a
decreased amount of multimeric forms of the substance compared to a
control lacking trehalose. Typically, trehalose prevents the
formation of all multimeric forms of the substance. In the case of
growth hormone, for instance, the addition of trehalose prior to
dehydrating or freezing results in the elimination of all
multimeric forms with the exception of dimers. The dimers are,
however, reduced in comparison to a control.
[0019] In a preferred embodiment, the substances suitable for use
in the invention have medical utility. Such substances include, but
are not limited to, therapeutic substances, prophylactic substances
and diagnostic substances. The substances are those which form
multimers upon dehydration/rehydration and/or freezing/thawing. The
method of formation of multimers or aggregates is not critical to
the invention.
[0020] Suitable therapeutic substances include, but are not limited
to, any therapeutically effective biological modifier. Such
modifiers include, but are not limited to, proteins and peptides,
steroid hormones, oligosaccharides, nucleic acids and a variety of
small molecules. Further, the-modifiers may be derived from natural
sources made by recombinant or synthetic means and include
analogues, agonists and homologs. As used herein "protein" refers
also to peptides and polypeptides. Such proteins include, but are
not limited to, growth hormones, growth factors, insulin,
monoclonal antibodies, interferons and interleukins. Preferably,
the growth hormone is human growth hormone. Suitable steroid
hormones include, but are not limited to, estrogen, progesterone
and testosterone. Therapeutic substances prepared by the methods
described herein are also encompassed by the invention.
[0021] Suitable prophylactic substances include, but are not
limited to, aluminum hydroxide and aluminum phosphate which are
used in preparation of vaccines. Compositions containing the
prophylactic substances are further encompassed by the invention.
Preferable compositions include vaccines containing the aluminum
hydroxide or aluminum phosphate prepared by the method described
herein. Suitable vaccines include, but are not limited to,
combination vaccines, such as diphtheria, tetanus, pertussis (DTP)
or DTP/inactivated poliovaccine (IPV). Suitable diagnostic
substances include, but are not limited to, colloidal gold,
polystyrene latex, fixed erythrocytes and monoclonal antibodies.
Diagnostic substances prepared by the method described herein are
also encompassed by the invention.
[0022] The dehydration step can be performed by any method known in
the art including, but not limited to, lyophilization, drying at
ambient conditions or drying under reduced vapor pressure. When
drying at reduced vapor pressure, the temperature at which the
drying occurs is preferably below the temperature at which
degradation of the substance occurs.
[0023] The freezing step can be performed by any method known in
the art including, but not limited to immersing in liquid nitrogen,
placing in a freezer which may be at -4.degree. C. to -80.degree.
C., dry ice and alcohol freezing bath. The samples should be
maintained at a temperature suitable to maintain the frozen state.
Thawing the frozen sample can be by any means known in the art, for
instance at room temperature or at an elevated temperature. If
thawing occurs at an elevated temperature, the temperature should
be below that which causes denaturation or other chemical changes
in the substance. Optimal freezing and thawing temperatures can be
determined empirically. Such a determination is within the skill of
one in the art.
[0024] Once the substances have been dehydrated or frozen, they can
be stored indefinitely. The dehydrated substances store well at
ambient temperatures, although they may be stored at any
temperature below that which causes denaturation or other chemical
changes. The invention further includes the steps of rehydration of
the dehydrated samples to obtain solutions and suspensions
substantially free of aggregates of the substance. Rehydration may
add at least an amount of water sufficient to restore the buffer
composition of the original solution or suspension but may add any
amount of water or buffer.
[0025] When the substance is red blood cells, the method may
further comprise fixing the red blood cells prior to adding
trehalose. The fixing step may be done by any method known in the
art including, but not limited to, glutaraldehyde. In the preferred
embodiment, the cells are fixed.
[0026] The methods of the present invention require that the
trehalose be present in an amount sufficient to prevent aggregation
of the substance upon rehydration or thawing. Such a determination
will be made empirically and is well within the skill of one in the
art. Preferably, trehalose is added in an amount to obtain a final
concentration of from about 1% to 50% (w/v). More preferably,
trehalose is added in an amount to obtain a final concentration of
from about 5% to 25% (w/v).
[0027] Trehalose is available from a variety of suppliers.
Preferably the grade of trehalose used is ANALAR reagent, molecular
biology or pharmaceutical grade. In the case of medicinal
compositions the trehalose preferably meets the good manufacturing
practice (GMP) standards set by the Food and Drug Administration
(FDA).
[0028] The invention also encompasses the products obtained by the
method both before and after rehydration or thawing. In one
embodiment, the invention includes the frozen compositions
containing a substance and an amount of trehalose sufficient to
prevent aggregation of the substance upon thawing. In another
embodiment, the invention includes a dehydrated composition
comprising a substance and an amount of trehalose sufficient to
prevent aggregation of the substance upon rehydration. The
invention further includes the compositions after being thawed or
rehydrated respectively.
[0029] Interestingly, the amount of trehalose found to be effective
at preventing aggregation cannot be directly extrapolated from the
amount of trehalose effective in preventing desiccation damage. For
instance, work presented in U.S. Pat. No. 4,891,319 showed that
amounts of trehalose as low as 1% w/v in a protein solution could
prevent desiccation damage to proteins such as Factor VIII. The
Examples presented herein show that more than 30% w/v trehalose is
necessary to completely prevent aggregation of aluminum hydroxide
and 15% w/v is necessary to prevent aggregation of a protein.
[0030] The following examples are meant to illustrate, but not
limit, the invention.
EXAMPLES
Example 1
Prevention of Aggregation of Particulate Suspensions by
Trehalose
[0031] In order to determine whether trehalose prevented
aggregation of particulate suspensions, two examples, colloidal
gold and polystyrene latex, were studied. Colloidal gold was
obtained from the Babraham Laboratories and polystyrene latex was a
suspension of particles of polystyrene which had been purchased
from Sigma Chemical Company.
[0032] The colloidal gold was made according to the method
described by Frens (1993) Nature 241:20. It was dried from a
concentrated suspension of 0.2% Au in a volume of 50 .mu.l per well
in a 96 well microtiter plate either with added 10% w/v trehalose
or without trehalose and subsequently rehydrated after storage for
one week at 37.degree. C. in a dry oven. On rehydration, the
material that had been dried in the presence of trehalose gave a
smooth suspension of colloidal gold as determined by microscopic
examination. The material that had been dried without trehalose
showed microscopic aggregates which could not be broken up into a
smooth suspension.
[0033] With the polystyrene latex, similar experiments were done.
The latex was obtained from Sigma Chemical Company catalogue number
LB-8, average diameter 0.8 micron polystyrene. It was used at the
concentration obtained from the supplier and again was dried
without any addition and also dried with the addition of 10% w/v
trehalose which was dissolved in the solution before drying. Both
samples were rehydrated about a week after drying and were stored
at 37.degree. C. in a dry oven in the interim. The material dried
without trehalose was badly aggregated into very large clumps. The
material dried in the presence of trehalose resuspended into a very
smooth, single particulate suspension.
[0034] Thus, the addition of trehalose prior to drying the
particulate suspensions substantially reduced the amount of
aggregation upon rehydration compared to a control lacking
trehalose.
Example 2
Effect of Trehalose-Drying on Aggregation of Red Blood Cells
[0035] Experiment
[0036] Rat RBCs were washed three times in an anti-coagulant CPD
(102 mM trisodium citrate, 1.08 mM sodium phosphate and 11 mM
dextrose), filtered through cotton wool and fixed in either 1%
formaldehyde or 0.5% glutaraldehyde. Fixing was at room temperature
for one hour. The fixed cells were washed three times in CPD and
resuspended in either 10% Trehalose and 0.12 mM Sodium Azide
(NaN.sub.3) or CPD. The final cell concentration was 25% w/v.
[0037] Cells fixed in formaldehyde lysed on washing and were not
processed further.
[0038] Unfixed cells agglutinated in trehalose and needed the
addition of 1/5th volume of Phosphate-buffered saline before being
processed further.
[0039] 100 .mu.l of cells in either 10% trehalose 0.12 mM NaN.sub.3
or CPD were dried either in Nunc plates or on slides and examined
microscopically for aggregates.
[0040] Results
[0041] The unfixed cells dried without trehalose lysed completely
and with those dried with trehalose also showed 95-99% lysis though
the ghosts showed discoid morphology.
[0042] The fixed cells dried without trehalose showed gross
macroscopic aggregation of the cells. The fixed cells dried with
trehalose resuspended as a smooth single cell suspension with only
a few microaggregates. These microaggregates appear to form at
higher concentrations of trehalose and thus do not appear to be
concentration dependent.
Example 3
Aggregation of Aluminum Hydroxide/Phosphate
[0043] Sedimentation Assay
[0044] The following method was followed to determine whether
trehalose is successful in preventing aggregation of prophylactic
adjuvants.
[0045] Aluminum phosphate and aluminum hydroxide were diluted
5-fold to a final concentration of 0.6% w/v and allowed to sediment
in 1 ml glass pipettes. The height of the sediment column was
measured at various time intervals up to 24 hours. Note that the %
height of sediment column should not be <30% when a steady state
has been reached. (about 5 hours.)
[0046] The samples were dried under vacuum, frozen at -20.degree.
C. and thawed at room temperature.
[0047] Results
[0048] Pilot 1. Aluminum Phosphate
[0049] Different forms of drying and storage were compared in the
presence or absence of 15% trehalose. These were vacuum drying
(Tv), freeze drying (Tfd), freezing (Tfz) and freeze thawing for
four cycles (T4fz). Wet controls (Tw) which were stored at
4.degree. C. were also run.
[0050] 200 .mu.l sample per glass vial were dried and sedimentation
assays carried out at day 0 and after 1 week storage at 45.degree.
C. The results obtained are shown in FIGS. 1 and 2.
[0051] Pilot 2. The aggregation of aluminum hydroxide and haemaccel
(degraded gelatin) was measured with a titration of trehalose with
the concentrations shown in Table 1. The samples contained 1.5%
aluminum hydroxide and 2% haemaccel. Only vacuum drying (d) and
freezing (f) were compared. Wet controls (w) contained trehalose
and haemaccel but were not dried or frozen. Each series contained
(d), (f) and (w) samples. The concentrations used are shown in
Table 1 and the results obtained are shown in FIG. 3.
1TABLE 1 Series Final % trehalose % haemaccel 1 7.5 -- 2 15 -- 3 30
-- 4 15 2 5 -- --
[0052] Conclusions
[0053] a) 15% trehalose can prevent freezing induced aggregation in
aluminum phosphate and aluminum hydroxide
[0054] b) 7.5% trehalose is not sufficient for preventing
aggregation during the drying process.
[0055] c) No additional effect of Haemaccel at 2% was observed.
[0056] Aluminum hydroxide, dried in the absence of trehalose and
rehydrated was found to be aggregated into large clumps which
sedimented rapidly and quickly to yield a very small gel column.
Trehalose in concentrations above 15% prevented this aggregation so
that the rehydrated material formed a gel column of a height
similar to the fresh, undehydrated material. This sedimentation
pattern illustrates that the hydrated, nonaggregated molecules have
a large hydration shell volume and are separated from one another
causing them to sediment slowly.
Example 4
Effect of Trehalose on Aggregation of Biological Molecules
[0057] Protein formulations may undergo modification by a number of
mechanisms including deamidation, oxidation and aggregation, the
principle causes of human growth hormone (hGH) degradation.
Deamidation and oxidation are considered collectively as chemical
degradation. To date there is little evidence of any effect of
these chemical degradation products on biopotency. Pearlman and
Bewly (1993) In: Wang and Pearlman eds. Stability and
Characterization of Protein and Peptide Drugs, pp. 1-58, Plenum
Press, New York.
[0058] Aggregation is the principle problem affecting hGH and other
protein formulations used as biopharmaceuticals and may reduce
biopotency. Soluble or insoluble aggregates can form as a result of
both covalent and non-covalent interactions. A variety of stresses
such as heating, freezing or agitation may induce aggregation.
Whilst a visible insoluble aggregate may render a parenteral
product unuseable, the major problem is the induction of an
unwelcome immune response in the subject. Pearlman and Bewley 1993.
This is particularly detrimental where the protein formulations
such as hGH are administered parenterally and in multiple
doses.
[0059] The following experiment was performed to determine whether
or not trehalose affected the aggregation of proteins. Samples of
hGH (5 mg) were dried from 200 .mu.l containing 15% trehalose, 5 mM
Na.sub.2HPO.sub.4-2H.sub.2O adjusted to pH 7.4 with H.sub.3PO.sub.4
(formulation A). Two control samples were prepared: 5 mg hGH dried
from 200 .mu.l sodium phosphate buffer pH 7.4 (formulation B); and
5 mg hGH dried from 200 .mu.l sodium phosphate buffer pH 7.4, 5 mg
glycine, 25 mg mannitol (formulation C). These formulations were
dried for 20 hours in a vacuum drier at a pressure of 30 millitorr
and a shelf temperature of 40.degree. C. They were subsequently
sealed under vacuum in standard pharmaceutical serum vials with
rubber closures and a crimped aluminum seal.
[0060] Following storage at 40.degree. C. in a dry incubator,
samples were rehydrated with deionised water and analysed by
reverse phase and size exclusion high performance liquid
chromatography to determine chemical degradation and aggregation
respectively according to the method described by Pikal et al.
(1991) Pharm. Res. 8:427-436. These results are presented in Table
2.
[0061] Formulation A was subsequently re-analysed and compared with
a conventionally freeze-dried essentially as described in Pikal et
al. (1991) equivalent formulation (formulation D). These results
are presented in Table 3.
[0062] Results
[0063] An accelerated aging protocol of four weeks at 40.degree. C.
was utilized to assess stability and aggregation. The formulation
containing trehalose performs very well under these conditions. No
chemical degradation was observed and the limited aggregation
detected was restricted to dimer formulation (Table 2, lines
1-4).
[0064] The absence of high molecular weight aggregates is
significant.
[0065] Two hGH controls were formulated, one without a stabilizing
excipient (B) and one containing glycine and mannitol that was
similar to commercial formulations (C) (Table 2, lines 5-6). These
formulations suffered from considerable chemical degradation and
aggregate formation, both dimer and higher molecular weight. The
values for the glycine mannitol formulation were comparable with
results from a previous study in which a similar formulation was
freeze-dried (Table 3, line 7, Pikal, et al. 1991). When the
stability of formulation A was compared with that of a freeze-dried
equivalent (formulation D), no difference in terms of 40.degree. C.
stability was observed (Table 3, lines 1-6). In Tables 2 and 3
chemical degradation is measured by the area under the curve
represented by the deamidated protein.
[0066] Thus the hGH formulations containing trehalose, either dried
at 40.degree. C. or freeze-dried, have been shown to be
considerable improvements on previous formulations.
2TABLE 2 Summary of hGH Stabilization and Aggregation Data (Part 1)
% % Chemical % Aggregation Formu- Degra- Aggregation High Line
lation Treatment dation Dimer Mol. Weight 1 A pre-dry 3.1 0.4 0.003
2 A post-dry 3.3 0.6 0.06 3 A 2 wk., 40.degree. C. 3.5 0.9 0.02 4 A
4 wk., 40.degree. C. 3.4 1.1 0.002 5 B 4 wk., 40.degree. C. 11.1
6.9 2.1 6 C 4 wk., 40.degree. C. 8.2 2.2 0.8
[0067]
3TABLE 3 Summary of hGH Stabilization and Aggregation Data (Part 2)
% Chemical Line Formulation Treatment Degradation % Aggregation 1 A
initial 4.15 0.66 2 A 2 wk., 40.degree. C. 4.16 0.92 3 A 4 wk.,
40.degree. C. 4.25 1.04 4 D initial 4.05 0.71 5 D 2 wk., 40.degree.
C. 4.09 0.86 6 D 4 wk., 40.degree. C. 4.17 0.92 7 E 4 wk.,
40.degree. C. 8.2 3.0
[0068] Although the foregoing invention has been described in some
detail by way of illustration and example for purposes of clarity
and understanding it will be apparent to those skilled in the art
that certain changes and modifications may be practiced. Therefore,
the description and examples should not be construed as limiting
the scope of the invention, which is delineated by the appended
claims.
* * * * *