U.S. patent application number 14/412786 was filed with the patent office on 2015-07-09 for annelid haemoglobin lyophilisation process.
The applicant listed for this patent is Hemarina. Invention is credited to Morgane Rousselot, Franck Zal.
Application Number | 20150190343 14/412786 |
Document ID | / |
Family ID | 48656247 |
Filed Date | 2015-07-09 |
United States Patent
Application |
20150190343 |
Kind Code |
A1 |
Zal; Franck ; et
al. |
July 9, 2015 |
ANNELID HAEMOGLOBIN LYOPHILISATION PROCESS
Abstract
The present invention relates to a lyophilizate comprising at
least one globin, one globin protomer or one extracellular
hemoglobin of annelids, and a stabilizer chosen from disaccharides,
polyols and antioxidants. The present invention also relates to a
composition comprising: a solution comprising at least one globin,
one globin protomer or one extracellular hemoglobin of annelids,
and a stabilizer chosen from disaccharides, polyols and
antioxidants. Finally, the present invention relates to a process
for preparing the lyophilizate.
Inventors: |
Zal; Franck;
(Ploujean-Morlaix, FR) ; Rousselot; Morgane;
(Saint Pol de Leon, FR) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Hemarina |
Morlaix |
|
FR |
|
|
Family ID: |
48656247 |
Appl. No.: |
14/412786 |
Filed: |
June 5, 2013 |
PCT Filed: |
June 5, 2013 |
PCT NO: |
PCT/FR2013/051271 |
371 Date: |
January 5, 2015 |
Current U.S.
Class: |
514/13.5 |
Current CPC
Class: |
A61K 38/1709 20130101;
A61K 47/26 20130101; A61K 38/1767 20130101; A61K 47/22 20130101;
A61K 35/62 20130101; A61K 9/19 20130101; A61P 7/08 20180101; A61K
38/42 20130101 |
International
Class: |
A61K 9/19 20060101
A61K009/19; A61K 47/26 20060101 A61K047/26; A61K 47/22 20060101
A61K047/22; A61K 38/17 20060101 A61K038/17 |
Foreign Application Data
Date |
Code |
Application Number |
Jun 5, 2012 |
FR |
12 55204 |
Claims
1. A lyophilizate comprising at least one extracellular hemoglobin,
globin protomer or globin of annelids, and a stabilizer chosen from
disaccharides, polyols and antioxidants.
2. A composition comprising: a solution comprising at least one
extracellular hemoglobin, globin protomer or globin of annelids,
and a stabilizing chosen from trehalose and ascorbic acid.
3. The lyophilizate of claim 1, wherein the extracellular
hemoglobin of annelids is an extracellular hemoglobin of polychaete
annelids.
4. The lyophilizate of claim 1, wherein the stabilizer is chosen
from trehalose, sucrose, mannitol, sorbitol and ascorbic acid,
preferably from trehalose and ascorbic acid.
5. A process for preparing the lyophilizate of claim 1, comprising:
i) the mixing of a solution comprising at least one extracellular
hemoglobin, globin protomer or globin of annelids with a stabilizer
chosen from disaccharides, polyols and antioxidants; ii) the
freezing of the mixture at a temperature of between -10.degree. C.
and -100.degree. C. for a time of at least 24 h; iii) the
sublimation of the frozen mixture for at least 2 h, under vacuum;
iv) the final drying of the mixture until a powder is obtained.
6. The process of claim 5, wherein the stabilizer comprises
disaccharides or polyols in a concentration of between 1 and 500
mg/ml.
7. The process of claim 5, wherein the stabilizer comprises
antioxidants in a concentration of between 3 and 20 mM.
8. The process of claim 5, wherein the stabilizer comprises
trehalose or sucrose present in a concentration of between 50 and
70 mg/ml, mannitol present in a concentration of approximately 50
to 100 g/l and ascorbic acid present in a concentration of
approximately 5 mM.
9. The process of claim 5, wherein the freezing is carried out at a
temperature of between -10.degree. C. and -90.degree. C., for at
least 24 h.
10. The process of claim 5, wherein the sublimation is carried out
for at least 4 h.
Description
[0001] The present invention relates to a process for the
lyophilization of at least one extracellular hemoglobin, globin
protomer or globin of annelids, and also to the lyophilisate
obtained.
[0002] The extracellular hemoglobin of annelids is of use as a
blood substitute, and makes it possible to treat problems
associated with an oxygen deficiency.
[0003] The term "blood substitute" is intended to mean any product
or solution which makes it possible to compensate for a blood loss
following a hemorrhage by providing oxygen carriers. A blood
substitute is different than an artificial blood, since the blood
substitute cannot perform all the functions provided by blood, such
as hormone transport for example.
[0004] Currently, two pharmaceutical classes of substitution
products are already known: they are perfluorocarbons (PFCs) and
hemoglobin oxygen carriers (HBOC).
[0005] PFCs are chemically synthesized compounds which allow the
transport of O.sub.2 in dissolved form in the bloodstream and to
the tissues.
[0006] HBOCs come from the purification and then chemical
modification of bovine HB or human HB (the latter coming from
expired blood bags) or are alternatively derived from synthesis by
genetic engineering.
[0007] In order to store HBOCs, a conventional method consists in
conditioning the hemoglobin molecules in solution. In order to
guarantee better stability, in some cases, it is necessary to store
them between -20.degree. C. and -80.degree. C. before their use.
They are then transported in dry ice in order to ensure that there
is no thawing. These various conditions involve an additional cost
and more restrictive storage and transportation logistics which can
prove to be limiting for certain applications.
[0008] There is therefore a need to have a functional hemoglobin
which is easily stored and transported, including in remote
environments without refrigeration.
[0009] Solutions exist, and one of them is lyophilization.
[0010] However, preliminary studies have revealed that the
hemoglobin molecules are partly degraded by the lyophilization
process, thereby in particular impairing their functionality,
namely their ability to reversibly bind oxygen. This is, moreover,
described in the literature for proteins in general (Heller Martin.
C, Carpenter John. F, Randolph Theodore. W, "Protein Formulation
and Lyophilization Cycle Design: Prevention of Damage due to
Freeze-Concentration Induced Phase Separation" Biotechnology and
Bioengineering, Vol 63, No. 2, 1999).
[0011] The inventors have discovered that, surprisingly, the
extracellular hemoglobin of annelids, when it is mixed with a
specific stabilizer, can be lyophilized, while at the same time
preserving its quaternary structure, its functionality and its
efficacy.
[0012] The present invention thus relates to a process for the
lyophilization of at least one extracellular hemoglobin, globin
protomer or globin of annelids. The present invention also relates
to the lyophilisate thus obtained. The latter in fact makes it
possible to store the extracellular hemoglobin of annelids, its
globin(s) and its globin protomer(s) in a format which is
economical and practical (i.e. it requires a minimum amount of
space), while at the same time preserving the structural and
functional properties of the hemoglobin.
[0013] The lyophilizate according to the invention comprises at
least one extracellular hemoglobin, globin protomer or globin of
annelids, and a stabilizer chosen from disaccharides, polyols and
antioxidants.
[0014] The present invention also relates to a composition
comprising: [0015] a solution comprising at least one extracellular
hemoglobin, globin protomer or globin of annelids, and [0016] a
stabilizer chosen from disaccharides, polyols and antioxidants,
preferably chosen from trehalose and ascorbic acid.
[0017] The extracellular hemoglobin of annelids is present in the
three classes of annelids: the polychaetes, the oligochaetes and
the achaetes. Reference is made to extracellular hemoglobin because
it is not naturally contained in a cell, and can therefore
circulate freely in the bloodstream without chemical modification
to stabilize it or make it functional.
[0018] The extracellular hemoglobin of annelids is a giant
biopolymer with a molecular weight of between 2000 and 4000 kDa,
consisting of approximately 200 polypeptide chains comprising
between 4 and 12 different types which are generally grouped into
two categories.
[0019] The first category, with 144 to 192 components, groups
together the "functional" polypeptide chains which bear an active
site of heme type, and are capable of reversibly binding oxygen;
these are chains of globin type, the weights of which are between
15 and 18 kDa and which are very similar to the .alpha.-and
.beta.-type chains of vertebrates.
[0020] The second category, with 36 to 42 components, groups
together the "structural" or "linker" polypeptide chains which have
few or no active site but enable the assembly of the subunits
called one-twelfth subunits or protomers.
[0021] Each hemoglobin molecule consists of two superposed hexagons
which have been named hexagonal bilayer and each hexagon is itself
formed by the assembly of six subunits (or "one-twelfth subunits"
or "protomers") in the form of a drop of water. The native molecule
is made up of twelve of these subunits (dodecamer or protomer).
Each subunit has a molecular weight of between 200 and 250 kDa, and
constitutes the functional unit of the native molecule.
[0022] Preferably, the extracellular hemoglobin of annelids is
chosen from the extracellular hemoglobins of polychaete annelids,
preferably from the extracellular hemoglobins of the family
Arenicolidae and the extracellular hemoglobins of the family
Nereididae. Even more preferentially, the extracellular hemoglobin
of annelids is chosen from the extracellular hemoglobin of
Arenicola marina and the extracellular hemoglobin of Nereis, more
preferentially the extracellular hemoglobin of Arenicola
marina.
[0023] According to the invention, the lyophilizate or the
composition may also comprise at least one globin protomer of the
extracellular hemoglobin of annelids. Said protomer constitutes the
functional unit of native hemoglobin, as indicated above.
[0024] Finally, the lyophilizate or the composition may also
comprise at least one globin chain of the extracellular hemoglobin
of annelids. Such a globin chain may in particular be chosen from
the Ax and/or Bx type globin chains of extracellular hemoglobin of
annelids.
[0025] The extracellular hemoglobin of annelids and globin
protomers thereof have an intrinsic superoxide dismutase (SOD)
activity, and the presence of antioxidant is not required in order
for them to function, contrary to the use of a mammalian
hemoglobin, for which the antioxidant molecules are contained
inside the red blood cell and are not bonded to the hemoglobin.
Furthermore, the extracellular hemoglobin of annelids, globin
protomers thereof and/or globins thereof do not require a cofactor
in order to function, contrary to mammalian hemoglobin, in
particular human hemoglobin. Finally, the extracellular hemoglobin
of annelids, globin protomers thereof and/or globins thereof do not
possess blood typing; they make it possible to avoid any problem of
immunological reaction.
[0026] The extracellular hemoglobin of annelids, globin protomers
thereof and/or globins thereof may be native or recombinant.
[0027] The composition according to the invention also comprises a
solution. This solution is capable of creating a saline environment
suitable for the hemoglobin, protomers thereof and globins thereof,
and thus makes it possible to maintain the quaternary structure,
and therefore the functionality of this molecule. By virtue of the
solution, the hemoglobin, protomers thereof and globins thereof are
capable of performing their oxygenation function.
[0028] The solution according to the invention is an aqueous
solution comprising salts, preferably chloride, sodium, calcium,
magnesium and potassium ions, and confer on the composition
according to the invention a pH of between 6.5 and 7.8; its
formulation is similar to that of a physiologically injectable
liquid. Under these conditions, the extracellular hemoglobin of
annelids, globin protomers thereof and globins thereof remain
functional.
[0029] In the present description, the pH is understood to mean at
ambient temperature (20.+-.5.degree. C.), unless otherwise
mentioned.
[0030] Preferably, the solution is an aqueous solution comprising
sodium chloride, calcium chloride, magnesium chloride, potassium
chloride, and also sodium gluconate and sodium acetate, and has a
pH of between 6.5 and 7.8, preferably equal to 7.1.+-.0.5,
preferably of approximately 7.35. More preferentially, the
stabilizing solution is an aqueous solution comprising 0-100 mM of
NaCl, preferably 90 mM of NaCl, 23 mM of Na gluconate, 2.5 mM of
CaCl.sub.2, 27 mM of Na acetate, 1.5 mM of MgCl.sub.2, 5 mM of KCl,
and has a pH of 7.1.+-.0.5, possibly containing between 0 and 100
mM of antioxidant of ascorbic acid and/or reduced glutathione type.
Said solution preferably has an osmolarity of between 300 and 450,
preferably between 300 and 350, and preferentially of 302
mOsmol/l.
[0031] The composition and the lyophilizate according to the
invention also comprise a stabilizer. This stabilizer maintains the
quaternary structure and therefore the functionality of the
hemoglobin, globins thereof and protomers thereof, even after
lyophilization.
[0032] The term "stabilizer" is intended to mean a disaccharide, a
polyol and/or an antioxidant. The disaccharides comprise in
particular sucrose, trehalose and raffinose, preferably trehalose.
The effectiveness of the stabilizer is determined by comparing the
physicochemistry and the functional properties of the hemoglobin
before and after lyophilization.
[0033] The stabilizer according to the invention is chosen from
disaccharides, polyols and antioxidants.
[0034] Preferably, the disaccharides are chosen from trehalose and
sucrose. More preferentially, the disaccharide is trehalose.
Preferably, the polyols are chosen from mannitol and sorbitol.
Finally, preferably, the antioxidant is ascorbic acid.
[0035] Trehalose is also called
.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside or
alpha,alpha-trehalose, or
.alpha.-D-glucopyranosyl-.alpha.-D-glucopyranoside dihydrate. It is
a disaccharide composed of two glucose molecules linked together by
a particularly stable .alpha.,.alpha.-1,1 (or
"1,1-.alpha.-glycosidic") bond.
[0036] Sucrose is a disaccharide formed by the condensation of a
glucose molecule with a fructose molecule. Its chemical name is
.beta.-D-fructofuranosyl-(2.revreaction.1)-.alpha.-D-glucopyranoside.
[0037] Mannitol, or 1,2,3,4,5,6-hexanehexol, and sorbitol, or
(2R,3S,4S,5S)-hexane-1,2,3,4,5,6-hexol, are polyols.
[0038] Finally, ascorbic acid is an organic acid which has
antioxidant properties. It may be present in D or L form.
Preferably, the stabilizer is L-ascorbic acid, or vitamin C.
[0039] Preferably, the stabilizer is chosen from trehalose and
ascorbic acid. Preferably, the composition and/or the lyophilizate
according to the invention comprise trehalose and ascorbic
acid.
[0040] The composition and the lyophilizate according to the
invention may comprise salts. These salts can be chosen from
sodium, calcium, magnesium and potassium salts. Preferably, the
salts are chosen from sodium chloride, calcium chloride, magnesium
chloride, potassium chloride, sodium gluconate and sodium
acetate.
[0041] A subject of the invention is also a process for preparing a
lyophilizate, comprising:
[0042] i) the mixing of a solution comprising at least one
extracellular hemoglobin, globin protomer or globin of annelids
with a stabilizer chosen from disaccharides, polyols and
antioxidants,
[0043] ii) the freezing of the mixture obtained in i) at a
temperature of between -10.degree. C. and -100.degree. C.,
preferably between -20.degree. C. and -100.degree. C., for a time
of at least 24 h, preferably at least 48 h;
[0044] iii) the sublimation of the frozen mixture obtained in ii)
for at least 2 h, under vacuum;
[0045] iv) the final drying of the mixture obtained in iii), until
a powder is obtained.
[0046] The mixing of step i) is carried out in particular by
vortexing.
[0047] Preferably, the stabilizer, in particular the disaccharide
or the polyol, is present in the mixture of step i) of the process
according to the invention in a concentration of between 1 and 500
mg/ml. Alternatively, the stabilizer, in particular the
antioxidant, is present in the mixture of step i) in a
concentration of between 3 and 20 mM. Even more preferentially, the
stabilizer present in the mixture of step i) is chosen from: [0048]
trehalose and sucrose which are present in a concentration of
between 50 and 70 mg/ml, preferably of approximately 55 mg/ml or 65
mg/ml, [0049] trehalose and sucrose which are present in a
concentration of approximately 100 mg/ml, [0050] mannitol which is
present in a concentration of between 50 and 100 g/l, and [0051]
ascorbic acid which is present in a concentration of approximately
5 mM.
[0052] The solution obtained at the end of step i) thus comprises
at least one extracellular hemoglobin, globin protomer or globin of
annelids, and the stabilizer.
[0053] This solution is subjected to lyophilization. The
lyophilization cycle may comprise three steps:
[0054] freezing (step ii) of the process according to the
invention):
[0055] This first phase consists in freezing the solution in such a
way that the water contained is converted into ice.
[0056] Preferably, the freezing of step ii) of the process
according to the invention is carried out at a temperature of
between -10.degree. C. and -100.degree. C., preferably between
-20.degree. C. and -90.degree. C., for at least 24 h, preferably at
least 48 h. Preferably, the freezing is carried out at
approximately -20.degree. C. for at least 24 h, preferably at least
48 h;
[0057] primary desiccation or sublimation (step iii) of the process
according to the invention):
[0058] The sublimation step allows the ice present in the frozen
solution to go from the solid state to the gas state, without an
intermediate step. The frozen solution is dried out by the
application of a vacuum; the ice then becomes vapor.
[0059] The sublimation is carried out using a high-vacuum pump, a
mechanical pump or a cryopump.
[0060] Preferably, the sublimation of step iii) is carried out for
at least 4 h;
[0061] secondary desiccation or final drying (step iv) of the
process according to the invention):
[0062] When the ice is totally sublimated, the secondary
desiccation phase can begin. It makes it possible to extract, by
desorption, the water molecules trapped at the surface of the dried
products.
[0063] At the end of the lyophilization, and therefore of the
process according to the invention, the lyophilizate obtained
comprises between 0.1% and 5% by weight of water.
[0064] The lyophilizate is a powder, which completely redissolves
in a hydrophilic liquid, without insoluble residues. The powder can
be stored in glass or plastic, preferably glass, bottles or
flasks.
[0065] The lyophilized hemoglobin thus obtained is easy to
transport and to store. The lyophilizate according to the present
invention is thus easy to reconstitute and is ready to be used.
[0066] The present invention also relates to a composition
comprising the lyophilizate according to the invention, and a
diluent. The lyophilizate can in fact be diluted at the appropriate
moment with a diluent, in order to restore the initial hemoglobin
solution. Preferably, the diluent is ultrapure water, so as not to
modify the concentration of the salts of the solution, and to
obtain a composition having a volume equivalent to that of before
the lyophilization.
[0067] The invention is described in greater detail in the
following examples. These examples are given solely for the
purposes of illustration, and are not limiting.
EXAMPLE 1
[0068] Materials and Methods
[0069] Study Protocol
[0070] The inventors evaluated the effects of the lyophilization
and the impact of the excipients added to the formation on: [0071]
the quaternary structure of the hemoglobins, [0072] the
functionality: ability to bind oxygen, [0073] the effectiveness of
the hemoglobins in cell models.
[0074] Hemoglobin (Hb) Preparation
[0075] The same batch of Hb was used for all the studies. A
calculated amount of Hb is thawed at 5.+-.3.degree. C. for 1 hour.
Once thawed, the required amount of excipient is added in liquid
form so as to achieve the desired concentration.
[0076] The solution thus obtained is frozen at -80.degree. C. for
at least 24 h. The frozen solution is then lyophilized in the
lyophilizer for approximately 4 h until a powder is obtained. This
powder is then stored at -80.degree. C. before the stability
study.
[0077] The reconstitution is carried out with ultrapure water so as
not to modify the salt concentration and in a volume equivalent to
that of before lyophilization.
[0078] Analytical Tools
[0079] The product monitoring study comprises three phases:
monitoring of the structure of the molecule, monitoring of its
functionality and, finally, monitoring of the hemoglobin
concentration.
[0080] Monitoring the Structure
[0081] The structure is monitored by means of a protein-specific
chromatography method: FPLC (Fast Protein Liquid Chromatography).
It uses the principle of size exclusion.
[0082] The system used is sold by Dionex.RTM. under the trade name
Ultimate 3000. It is entirely automated and controlled by the
accompanying Chromeleon.RTM. software.
[0083] The columns used are sold by GE Healthcare.RTM. and are of
the Superose 6, 10.times.300 mm type; they make it possible to
separate molecules of which the molecular weight is between 5 MDa
and 5000 Da in one hour at a flow rate of 0.5 ml/min The separation
is carried out under isocratic conditions, i.e. there is just one
elution buffer.
[0084] The acquisition in the context of the hemoglobin studies is
carried out at 2 wavelengths (280 nm and 414 nm). In order to
detect the target molecule, hemoglobin, the optical density is
measured at 280 nm (protein absorption peak) and at 414 nm (heme
absorption peak). The acquisition at 414 nm makes it possible to
identify the hemoglobin among the other proteins observed at 280 nm
At this same wavelength, it is also possible to monitor the
hemoglobin dissociation kinetics. The Chromeleon.RTM. software
provided with the equipment makes it possible both to harvest the
data and also to process them.
[0085] The data processing is carried out by analysis of the
chromatograms obtained at 280 nm and at 414 nm.
[0086] The software makes it possible to integrate the
chromatograms: [0087] the area under the curve of the peak of
interest is proportional to the concentration; [0088] the relative
percentage purity of each peak calculated by the software makes it
possible to monitor the evolution of the degradation of the
molecule over time.
[0089] In the case of the study, kinetics making it possible to
quantify the effects of the excipients selected and the behavior of
the molecule over the course of one week are performed. For this,
the chromatograms of each excipient are integrated at each point
(from time T.sub.0 up to day 5) and then the data generated by the
software (the area under the curve and the relative percentage
purity) are processed graphically using the Graphpad.RTM. software
in order to determine the degradation kinetics and to compare
them.
[0090] Monitoring of the Functionality
[0091] The functionality of the hemoglobin molecule is defined by
its ability to reversibly bind oxygen. This study is possible with
UV-visible spectrophotometry.
[0092] Hemoglobin has a characteristic spectrophotometric signature
which changes according to its functionality and its oxidation
state. The measurement is carried out on a window of wavelengths of
between 250 and 700 nm. Thus, absorption spectra which are
different according to the oxidation state are obtained.
[0093] For example, the hemoglobin of Arenicola marina (HbAm)
exhibits, in its oxygenated state, two peaks in the visible range,
i.e. the alpha and beta bands respectively present at 576 nm and
540 nm, and a "Soret" band at 414 nm. The first mentioned are
characteristic of the absorption of the complex formed between the
heme group, iron and oxygen. The Soret band is, for its part,
synonymous with the absorption of the complex formed between the
polypeptide chain and the heme group.
[0094] Hemoglobin has spectral properties characteristic of its
changes in conformation between the oxygenated and nonoxygenated
states.
[0095] Thus, depending on the maxima noted, the various states of
the hemoglobin can be determined by referring to the following
table:
TABLE-US-00001 Soret band Alpha band Beta band Hemoglobin form 414
.+-. 2 nm 540 .+-. 2 nm 576 .+-. 2 nm Oxyhemoglobin 430 .+-. 2 nm
555 .+-. 2 nm 555 .+-. 2 nm Deoxyhemoglobin 418 .+-. 2 nm 535 .+-.
2 nm 570 .+-. 2 nm Carboxyhemoglobin 406 .+-. 2 nm 500 .+-. 2 nm
630 .+-. 2 nm Methemoglobin
[0096] Finally, the percentage of oxidized hemoglobin can be
determined The UV-visible spectra acquired between 250 and 700 nm
are standardized at 523 nm (isobestic point). The absorbances at
576 nm (alpha band) and 540 nm (beta band) are added for each
spectrum. Likewise, the absorbances for a reference sample ideally
not exhibiting oxidation (in the case of lyophilization=the D0 of a
non-lyophilized sample is chosen as reference) are noted. The same
operation is then carried out for a sample considered to be 100%
oxidized (obtained by incubation at 37.degree. C. until
disappearance of the alpha and beta bands).
[0097] The percentage oxidation of the molecule is obtained by
means of the following calculation:
% Hb oxidized = 100 - [ A IIb - A 100 ] .times. 100 A 0 - A 100
##EQU00001##
[0098] With A=A.sub.540+A.sub.576; A Hb=A.sub.540+A.sub.576 of the
hemoglobin sample; A0=A.sub.540+A.sub.576 of the 0% oxidized; A
100=A.sub.540+A.sub.576 of the 100% oxidized.
[0099] As for the monitoring of the structure, the spectra are
superposed for each condition in order to visualize the effect of
the excipients over the course of one week, and then the evolution
of the oxidation percentages is processed graphically using the
Graphpad.RTM. software in order to determine the auto-oxidation
kinetics under the various conditions tested.
[0100] Monitoring the Concentration
[0101] The hemoglobin is assayed by spectrophotometry using
Drabkin's reagent which makes it possible to accurately assay the
heme, which is the active side of the hemoglobin.
[0102] Effectiveness Tests
[0103] In order to evaluate the effectiveness of the hemoglobins of
Arenicola marina (HbAm) and of Nereis (HbN), two cell models were
developed in the laboratory, each representative of the
applications to which the molecules are dedicated.
[0104] For HbAm, this involves a cell model which mimics the
storage conditions for organs awaiting transplantation. A cell
toxicity test is used in order to evaluate the lesions caused by
the cold storage: this test corresponds to the release of lactate
dehydrogenase (LDH). LDH is an enzyme present in the cytosol of
cells and its release into the culture supernatant reflects plasma
membrane permeabilization and therefore cell death. The
effectiveness of the HbAms is evaluated by comparing the percentage
LDH release under the organ storage conditions with and without
HbAm.
[0105] The second model developed is based on the application of
the HbN molecule in the context of the bioproduction of recombinant
proteins. The cell line corresponds to a line commonly used for
bioproduction. The cells are seeded at a predetermined cell density
in the presence or absence of a known amount of HbN. After 4 days
of culture, the cell density and the cell viability are measured.
The effectiveness of the HbNs is evaluated by comparing the cell
growth and viability under the culture conditions with and without
HbN.
[0106] Results for HbAm
[0107] Monitoring of the stability of HbAm after reconstitution
(T.sub.0)
[0108] Monitoring of the Hemoglobin Concentration after
Reconstitution
[0109] The results of the HbAm assay after reconstitution following
the lyophilization step indicate a decrease in the Hb concentration
after reconstitution, whatever the lyophilization conditions (see
FIG. 1: legend: NL=Non-lyophilized; L=Lyophilized; L+T=Lyophilized
with trehalose; L+Aa=Lyophilized with ascorbic acid;
L+M=Lyophilized with mannitol; L+S=Lyophilized with sucrose). This
loss of less than 10% indicates that the lyophilization decreases
the hydrophilic capacity of the molecule.
[0110] Monitoring of the Structural Stability at T0
[0111] The HbAm purity percentages at 280 nm and at 414 nm are
similar for the various compositions tested.
[0112] In the light of the standard deviations, the results
obtained show that there is no significant difference in purity
between the various conditions tested. Thus, at T0, after
reconstitution, the quaternary structure of the HbAm is not
significantly affected, either by the lyophilization or by the
presence of the various excipients.
[0113] Monitoring of the Functionality of Reconstituted HbAm
[0114] In order to judge as accurately as possible the impact of
the lyophilization on the functionality of the molecule after
reconstitution (T.sub.0), the percentage oxidation and the
modifications of the spectrophotometric signature of the hemoglobin
are measured.
[0115] The lyophilization process causes an oxidation of the
molecule of approximately 20% compared with the non-lyophilized
molecule. All the excipients tested, and in particular the ascorbic
acid and the trehalose, show a positive effect on the protection of
the molecule against oxidation.
[0116] The trehalose and the ascorbic acid made it possible both to
protect the molecule against the oxidation induced by the
lyophilization process and also to reduce the proportion of
molecule initially oxidized.
[0117] The study of the UV--visible spectra shows that the various
conditions tested do not significantly affect the functionality of
HbAm after reconstitution: the Soret, alpha and beta bands are
present at the wavelengths of a functional hemoglobin (cf. table
1). In the case of the hemoglobin lyophilized alone, although 20%
oxidation is observed, the molecule remains functional overall.
TABLE-US-00002 TABLE 1 Wavelengths noted on the UV-visible spectra
of the various conditions Alpha Beta Conditions Soret band band
band Functionality HbAm NL 414 nm 540 nm 574 nm Oxyhemoglobin HbAm
L 412 nm 538 nm 574 nm HbAm L + Trehalose 414 nm 538 nm 574 nm HbAm
L + Mannitol 412 nm 538 nm 574 nm HbAm L + Sucrose 414 nm 538 nm
574 nm HbAm L + Ascorbic 414 nm 540 nm 574 nm acid NL =
Non-lyophilized L = Lyophilized
[0118] Conclusions
[0119] This preliminary step shows that the lyophilization of HbAm
does not significantly modify the quaternary structure of the
molecule. However, the lyophilization causes a partial oxidation of
the hemoglobin. The results show that the excipients tested make it
possible to protect the molecule from the oxidation induced by the
lyophilization process without affecting its quaternary structure.
Moreover, among the excipients tested, trehalose and ascorbic acid
give the best protection against oxidation.
[0120] Monitoring of the Stability of HbAm Over the Course of 5
Days
[0121] This study is carried out on the samples reconstituted after
lyophilization with and without excipients and compared to the
non-lyophilized molecule.
[0122] Said samples are reconstituted at T.sub.0 with ultrapure
water and then incubated at 37.degree. C. so as to accelerate the
degradation of the molecule in order to evaluate the effects of the
chosen excipients. The structural and functionality analyses and
also the assaying of the hemoglobin are carried out each day
(D.sub.0, D.sub.1, D.sub.2, D.sub.3, D.sub.4).
[0123] Monitoring of the Hemoglobin Concentration
[0124] The results show a relative decrease in the amount of
hemoglobin. This decrease, characteristic of a degradation or
adsorption of the hemoglobin, is observed for the non-lyophilized
molecule, and also the molecule lyophilized in the presence of
ascorbic acid, mannitol and sucrose. No significant decrease for
the molecule lyophilized alone and in the presence of trehalose is
observed.
[0125] Monitoring of the Quaternary Structure
[0126] The percentage purity is measured at 280 nm over time. The
processing of the data (Graphpad.RTM. software) makes it possible
to determine a trend curve characteristic of the molecule
degradation kinetics. Two trends stand out: [0127] a linear
regression for all the conditions except trehalose; [0128] a
plateau followed by a decrease phase for trehalose.
[0129] The results show that ascorbic acid appears to maintain the
structural integrity of the HbAm molecule.
[0130] Specifically: [0131] the least stable molecule is the
molecule lyophilized alone: T.sub.1/2=2.9 days and k.sub.d=0.17
d.sup.-1; and [0132] the molecule lyophilized in the presence of
trehalose or of ascorbic acid is the most stable with T.sub.1/2=5.4
and 4.7 days respectively, and k.sub.d=0.12 and 0.10-1
respectively.
[0133] Monitoring of the Functionality
[0134] As for the monitoring of the structure, the oxidation data
were analyzed with Graphpad.RTM. in order to determine the molecule
oxidation kinetics.
[0135] The evolution of the percentage oxidation over time can be
represented by a sigmoid curve of the dose-response type for 3
conditions: lyophilized alone; lyophilized with trehalose and
sucrose.
[0136] The dose-response aspect can be explained by double
kinetics: the first corresponding to the auto-oxidation of the
molecule: the conversion of the ferrous iron to ferric iron and the
formation of radical O.sub.2 (O.sub.2..sup.-). Radical O.sub.2 is
an oxidizing species which catalyses the oxidation reaction
(equation below).
Hb(Fe.sup.2+)+O.sub.2+H.sub.2O.fwdarw.HB(Fe.sup.3+)OH.sub.2+O.sub.2..sup-
.-
[0137] Ascorbic acid and trehalose "capture" O.sub.2..sup.-,
preventing its interaction with Hb: this explains the linear
profile of the oxidation kinetics in the presence of these
excipients.
[0138] The study of the UV-visible spectra (results not shown)
shows that the functionality of the hemoglobin is maintained up to
D3 for the samples composed of ascorbic acid and up to D2 for those
containing trehalose.
[0139] Conclusions
[0140] During this short-term study, the predominance of two
excipients was observed for maintaining the structure and the
functionality and for slowing down the oxidation of the molecule
once reconstituted; these are trehalose and ascorbic acid.
[0141] Evaluation of the Effectiveness of HbAm
[0142] The results show that, for all the conditions tested,
compared to the control, i.e. to the preserving medium alone, the
LDH release percentages are lower than those of said medium.
[0143] This shows that HbAm is effective whatever the conditions.
However, it is more or less effective depending on the excipients
used during the lyophilization.
[0144] A significant difference is noted between the
non-lyophilized molecule and the molecule lyophilized in the
presence of ascorbic acid, which is the most effective. Finally,
the lyophilized molecule appears to be more effective than the
non-lyophilized molecule and than the molecule
lyophilized+trehalose.
[0145] Study of the Stability of HbAm in the Lyophilized State
[0146] The results presented here are very preliminary
[0147] Monitoring of the Hemoglobin Concentration
[0148] The evolution of the concentration over time under the
various conditions studied shows that there are no significant
differences up to D.sub.2. From D.sub.2 onward, the redispersion
appears to be more difficult.
[0149] Monitoring of the Structural Stability
[0150] The results obtained for the monitoring of the quaternary
structure of the molecule show surprising profiles in the case of
the molecule treated with ascorbic acid.
[0151] The plots could not be integrated starting from D.sub.1.
This is because the peak corresponding to the hemoglobin is
destructured.
[0152] A comparison between the purity of the molecule lyophilized
alone or with trehalose was carried out.
[0153] In the case of the lyophilized molecule, the curve describes
a linear trend (R.sup.2=0.9858). The molecule supplemented with
trehalose appears to be more stable. Indeed, the profile of the
curve shows a slight decrease between D.sub.0 and D.sub.1 and then
a stability phase up to D.sub.3.
[0154] Monitoring of the Functional Stability
[0155] In the light of the evolution of the percentage oxidation,
ascorbic acid and trehalose also appear to be effective. The first
describes an increase in linear evolution, while the second
exhibits a slight increase and then a stability phase. These
experiments will have to be reproduced in order to confirm these
first observations.
[0156] Results for HbN
[0157] All the experiments carried out in the context of the
monitoring of HbAm stability are applied to HbN.
[0158] Monitoring of the Stability of HbN at T.sub.0
[0159] Monitoring of the Hemoglobin Concentration at T.sub.0
[0160] The results of the hemoglobin assay after reconstitution of
the HbN molecule under the various conditions do not show any
significant difference between the non-lyophilized molecule, the
molecule lyophilized+trehalose and the molecule
lyophilized+mannitol. However, a loss of hemoglobin after
lyophilization of the molecule alone, supplemented with sucrose or
supplemented with ascorbic acid is noted.
[0161] In the present case both mannitol and trehalose, have a
positive effect on reconstitution.
[0162] Monitoring of the Structural Stability of HbN after
Reconstitution
[0163] The percentage purity measured at T.sub.0 at 280 nm (A) and
414 nm (B) does not, in the light of the standard deviations, show
any significant difference at 280 nm, whatever the state of the
molecule (lyophilized or non-lyophilized) and the excipients
tested. The same is true for the percentage purity at 414 nm. Thus,
the lyophilization does not appear to induce destructuring of the
molecule.
[0164] Monitoring of the Functionality of the HbN Molecule at
T0
[0165] The relative percentage oxidation obtained after
lyophilizationn and calculated on the basis of the non-lyophilized
molecule is measured.
[0166] The monitoring of the oxidation state of the molecule at
T.sub.0 shows an oxidation of 35% for the molecule lyophilized
alone, i.e. 15% higher than the HbAm molecule. All the excipients
tested provide the hemoglobin with protection against oxidation.
Specifically, the percentage oxidation noted at T.sub.0 is between
10% for trehalose and -20% for ascorbic acid which is the most
protective. The effect of trehalose is less effective for HbN than
for HbAm. The UV-visible spectra of the various samples exhibit
perfect superposition with a spectral signature of a functional
hemoglobin.
[0167] Thus, the functionality of the HbN molecule, like HbAm, is
not significantly affected by the lyophilization process.
[0168] Conclusions
[0169] At the end of this step, all the excipients tested maintain
the structure of the molecule and its functionality and also
provide the molecule with effective protection against oxidation,
particularly with a treatment with sucrose or with ascorbic acid.
Unlike in the case of HbAm, trehalose provides less effective
protection against oxidation. Indeed, in the case of HbN, the
oxidation is reduced (10%), but not obliterated.
[0170] The action of all the excipients tested will therefore be
evaluated over the course of 5 days in order to select the most
effective thereof.
[0171] Monitoring of the Stability of Reconstituted HbN Over the
Course of 5 Days
[0172] Monitoring of the Hemoglobin Concentration
[0173] The evolution of the hemoglobin concentration over time
shows that, whatever the condition considered, the hemoglobin
concentration remains stable.
[0174] Monitoring of the Quaternary Structure
[0175] The evolution of the percentage purity at 280 nm shows that
the various kinetics adopt a linear trend (table 2).
TABLE-US-00003 TABLE 2 HbN trend curves and associated constants
(NL = Non-lyophilized HbN; L = Lyophilized HbN) Associated
constants Condition Trend R.sup.2 tested curve Equation Half-life
T.sub.1/2(d) k.sub.d(d.sup.-1) NL 0.9755 0.1643 .+-. 0.01502 3.043
L 0.9874 0.1714 .+-. 0.01119 2.917 L + Trehalose 0.9737 0.1820 .+-.
0.01727 2.747 L + Ascorbic acid Linear regression % purity ( t ) %
purity ( t 0 ) = - k .times. t ##EQU00002## 0.9952 0.04049 .+-.
0.001995 12.348 L + Mannitol 0.9719 0.03847 .+-. 0.004627 12.997 L
+ Sucrose 0.9539 0.03423 .+-. 0.005324 14.607
[0176] The data show maintenance of the protein structure provided
by sucrose (T.sub.1/2=14.607; k.sub.d=0.03423), mannitol
(T.sub.1/2=12.997; k.sub.d=0.03847) and ascorbic acid
(T.sub.1/2=12.348; k.sub.d=0.0409). With regard to trehalose
(T.sub.1/2=2.747; k.sub.d=0.1820), it lies at the same level as the
lyophilized or non-lyophilized samples.
[0177] Monitoring of the Functionality
[0178] The monitoring of the percentage oxidation of the hemoglobin
reveals a protective effect provided by trehalose up to D4 with a
percentage oxidation of less than 50%. The other excipients tested
show an antioxidant effect at T0 which they gradually lose over the
days which follow, until they reach the oxidation level of the
non-lyophilized control. The profile of the curves adopts a
hyperbolic trend. The oxidation of the molecule is then very
quickly catalyzed by radical oxygen. The study of the UV-visible
spectra obtained for the conditions tested shows that the
functionality is maintained up to D3 when the hemoglobin is treated
with trehalose (results not shown here).
[0179] Conclusions
[0180] At the end of this monitoring over the course of one week,
two excipients stand out: trehalose for maintaining the
functionality and effective protection against oxidation; ascorbic
acid for maintaining the structure of the molecule. Another
excipient could be conjugated to trehalose; said other excipient is
sucrose. In order to complete this study, the tests for
effectiveness of the molecule formulated with these excipients will
be carried out.
[0181] HbN Effectiveness Evaluation
[0182] The HbN effectiveness test is carried out on a cell model.
The measurements carried out concern the percentage viability
(percentage of live cells) and the cell density.
[0183] The results show that the hemoglobin supplemented with
various excipients remains effective on the cells. Indeed, the
cells remain more than 95% viable and the cell density is
significantly increased compared with the control (.times.1.7 on
average).
[0184] A greater effectiveness is observed for the molecule
lyophilized in the presence of ascorbic acid and also for the
molecule in its lyophilized state without excipient.
[0185] Trehalose, for its part, describes a profile similar to that
of the non-lyophilized molecule.
[0186] Study of the Stability of HbN in the Lyophilized State
[0187] For this study, it is necessary to maintain the product
lyophilized in the presence or absence of excipients at 37.degree.
C. in order to accelerate the degradation of the molecule. It is
then reconstituted in water in order to carry out the analyses for
monitoring structure, function and concentration.
[0188] Monitoring of the Hemoglobin Concentration
[0189] The monitoring of the hemoglobin concentration after
reconstitution shows the same profile already observed for HbAm.
Specifically, no significant difference can be observed up to D2
before beginning a decrease phase synonymous with difficult
redispersion.
[0190] Monitoring of the Quaternary Structure
[0191] In the same way as for HbAm, the profiles of the
chromatograms are surprising for ascorbic acid in particular with
destructuring of the peak characteristic of hemoglobin.
[0192] Monitoring of the Functionality
[0193] In the case of HbN, the molecule lyophilized alone without
excipient is very strongly oxidized. When the effect of the chosen
excipients is studied, trehalose provides protection against
oxidation which is stable from D1 to D3. The same is true for
ascorbic acid, with a greater protective effect. The profile
obtained with a slight increase and then a stability phase is
similar to that of HbAm. There is despite everything a significant
difference between the two molecules in terms of the initial and
final percentage oxidation, twice as high for HbN.
EXAMPLE 2
[0194] Samples of 20 ml of HbAm having the following composition
were lyophilized: [0195] Sample A (control): HbAm in ultrapure
water; [0196] Sample B: HbAm in an aqueous 5 mM ascorbic acid
solution; [0197] Sample C: HbAm in an aqueous solution comprising
55 mg/ml of trehalose; [0198] Sample D: HbAm in an aqueous solution
comprising 55 mg/ml of trehalose and 5 mM of ascorbic acid.
[0199] These lyophilized samples were redispersed in i) ultrapure
water, ii) an aqueous 5 mM ascorbic acid solution, iii) a
stabilizing solution (=aqueous solution comprising 90 mM of NaCl,
23 mM of Na gluconate, 2.5 mM of CaCl.sub.2, 27 mM of Na acetate,
1.5 mM of MgCl.sub.2, 5 mM of KCl, pH of 7.1.+-.0.5) or iv) the
solution iii) additionally comprising 5 mM of ascorbic acid.
[0200] The functionality of the redispersed lyophilizates obtained
was measured as indicated in example 1.
[0201] The results show that the presence of trehalose during the
formulation limits the degradation of HbAm. The percentage of
hemoglobin in the redispersed samples C and D (i.e. with
trehalose), of around 88%, is significantly higher than that of the
samples A and B, of around 82-84%.
[0202] In addition, the presence of ascorbic acid during the
formulation limits the oxidation associated with the
lyophilization.
[0203] Lyophilization experiments were carried out with a sample E.
This sample E comprises HbAm in an aqueous solution comprising 23
mM of Na gluconate, 2.5 mM of CaCl.sub.2 , 27 mM of Na acetate, 1.5
mM of MgCl.sub.2, 5 mM of KCl, pH of 7.1.+-.0.5, 65 mg/ml of
trehalose and 5 mM of ascorbic acid.
[0204] It was possible to carry out the lyophilization very
satisfactorily.
EXAMPLE 3
[0205] Material:
[0206] Labconco laboratory lyophilization apparatus
[0207] Methods:
[0208] 1 ml aliquots of HbAm (M101) or HbN (M201)
[0209] The 1 ml samples are frozen before lyophilization and
dispensed into vials as indicated in table 1 below:
TABLE-US-00004 TABLE 1 Summary of the conditions tested. The
conditions E and F are positive controls for lyophilization
Reference Content Freezing T A M101 -20.degree. C. B M101
-80.degree. C. C M201 -20.degree. C. D M201 -80.degree. C. E 5%
Mannitol -20.degree. C. F 5% Mannitol -80.degree. C.
[0210] Once frozen, the vials are placed in the lyophilizer.
[0211] Results:
[0212] Lyophilization Results:
[0213] After verification, the -20.degree. C. freezer showed an
actual temperature of -17.degree. C.
[0214] After loading, the apparatus is pressurized, the samples A
and B leave the vial as a foam. The sample C shows limited partial
fusion. The other samples, which are in the "ice" zone of the vapor
pressure diagram, remain stable. After 30 minutes, the samples D, E
and F begin a sublimitation apparently under good conditions but
outside control. After operating for 6 hours, the test is stopped.
The samples D, E and F appear as an excellent chip, with
crystallization clearly apparent. The sample D has a shiny surface
suggesting the formation of a skin due to the freezing. The
redispersion takes place in a few minutes, but a few "lumps" are
present in the sample E.
[0215] Results of the HPLC Analyses:
[0216] HPLC analyses were carried out in order to verify the purity
and the structure of the hemoglobins post-lyophilization and in
order to verify whether said hemoglobins were not degraded during
the lyophilization.
TABLE-US-00005 Samples % purity (%) Clusters (%) Dissociation (%)
M101 Before Lyoph 91.80 4.01 1.49 M101 Post Lyoph 91.69 3.15 1.27
M201 Before Lyoph 86.55 5.28 2.81 M201 Post Lyoph 82.32 5.60
4.39
[0217] Results of the Hb Assays:
[0218] Hemoglobin assays were carried out before and after
lyophilization, and once dispersed.
TABLE-US-00006 Before lyophilization After lyophilization Samples
Molecules (mg/ml) (mg/ml) E M201 46.83 40.06 F M101 49.05 41.87
[0219] Conclusion:
[0220] The first conclusion is that the 2 molecules are
lyophilizable, since excellent chips were obtained for the
conditions at -80.degree. C. Indeed, the freezing at -20.degree. C.
results in the fusion of the molecules during the application of a
vacuum. The sublimation takes place correctly, but, given the
duration of the manipulation, there was virtually no secondary
desiccation, which, if extended, could only improve in particular
the moisture-content and redispersion conditions. The results of
the HPLC analyses show that the M201 molecule undergoes a partial
degradation (.about.4%) during the lyophilization. As regards the
M101 molecule, it is stable.
* * * * *