U.S. patent application number 16/294506 was filed with the patent office on 2019-08-22 for stabilized antibody compositions.
This patent application is currently assigned to NOVALIQ GMBH. The applicant listed for this patent is NOVALIQ GMBH. Invention is credited to Gesche Graf, Bernhard Gunther, Anthony Pettigrew, Dieter Scherer.
Application Number | 20190256591 16/294506 |
Document ID | / |
Family ID | 48803483 |
Filed Date | 2019-08-22 |
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United States Patent
Application |
20190256591 |
Kind Code |
A1 |
Gunther; Bernhard ; et
al. |
August 22, 2019 |
STABILIZED ANTIBODY COMPOSITIONS
Abstract
The invention provides novel compositions of antibodies based on
liquid vehicles selected from semifluorinated alkanes. The use of
these vehicles provides for improved stability and shelf-life of
antibodies and their derivatives. The compositions are useful for
topical administration or for parenteral injection.
Inventors: |
Gunther; Bernhard;
(Dossenheim, DE) ; Scherer; Dieter; (Laufen,
CH) ; Pettigrew; Anthony; (Heidelberg, DE) ;
Graf; Gesche; (Bensheim, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
NOVALIQ GMBH |
Heidelberg |
|
DE |
|
|
Assignee: |
NOVALIQ GMBH
Heidelberg
DE
|
Family ID: |
48803483 |
Appl. No.: |
16/294506 |
Filed: |
March 6, 2019 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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14907254 |
Jan 22, 2016 |
10273298 |
|
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PCT/EP2014/065840 |
Jul 23, 2014 |
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16294506 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/55 20130101;
A61P 43/00 20180101; A61P 9/14 20180101; A61P 37/06 20180101; C07K
2317/626 20130101; C07K 2317/569 20130101; C07K 2317/94 20130101;
C07K 2319/00 20130101; A61P 37/00 20180101; A61K 39/39591 20130101;
C07K 2317/21 20130101; A61K 39/395 20130101; C07K 2317/24 20130101;
C07K 16/28 20130101; A61P 35/00 20180101; C07K 2317/76 20130101;
C07K 2317/622 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; A61K 39/395 20060101 A61K039/395 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 23, 2013 |
EP |
13177699.9 |
Claims
1. Composition comprising an antigen-binding polypeptide or protein
and a liquid vehicle, wherein the liquid vehicle comprises a
semifluorinated alkane of the formula RFRH wherein RF is a linear
perfluorinated hydrocarbon segment with 4 to 12 carbon atoms, and
wherein RH is a linear alkyl group with 4 to 8 carbon atoms; and
wherein the antigen-binding polypeptide or protein is incorporated
in the composition such as to form a dispersion or suspension.
2. The method according to claim 10, wherein the antigen-binding
polypeptide or protein is selected from a monoclonal antibody,
polyclonal antibody, an antibody fragment, a fusion protein
comprising an antibody fragment, an antibody-drug conjugate, or any
combination thereof.
3. The method according to claim 2, wherein the antibody fragment
is a fragment antigen-binding (Fab), a single-chain variable
fragment (scFv), a single-domain antibody, a minibody, or a
diabody.
4. The method according to claim 2, wherein the monoclonal antibody
is a chimeric, humanized, or human antibody.
5. The method according to claim 10, wherein the antigen-binding
polypeptide or protein has a molecular mass of at least 90 kDa.
6. The method according to claim 10, wherein the semifluorinated
alkane is selected from F4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and
F6H10.
7. The method according to claim 10, wherein the composition is
substantially free of water.
8. The method according to claim 10, wherein the composition
retains at least 85% of its initial antigen-binding activity during
storage of 6 months at a temperature of between RT to 40.degree.
C.
9. The method according to claim 10, wherein the antigen-binding
polypeptide or protein is at a concentration of at least 0.5
mg/mL.
10. A method for treating or preventing a disease or condition in a
patient in need thereof, comprising the step of administering to
the patient an effective amount of a composition comprising an
antigen-binding polypeptide or protein and a liquid vehicle,
wherein the liquid vehicle comprises a semifluorinated alkane of
the formula RFRH wherein RF is a linear perfluorinated hydrocarbon
segment with 4 to 12 carbon atoms, and wherein RH is a linear alkyl
group with 4 to 8 carbon atoms; and wherein the antigen-binding
polypeptide or protein is incorporated in the composition such as
to form a dispersion or suspension.
11. The method according to claim 10, wherein the composition is
administered by subcutaneous, dermal, intramuscular, or
locoregional injection.
12. A method of stabilizing an antigen-binding polypeptide or
protein, comprising the step of mixing the antigen-binding
polypeptide or protein with a liquid vehicle comprising a
semifluorinated alkane of the formula RFRH wherein RF is a linear
perfluorinated hydrocarbon segment with 4 to 12 carbon atoms, and
wherein RH is a linear alkyl group with 4 to 8 carbon atoms, such
as to form a suspension or a dispersion.
13. The method of claim 12, wherein the antigen-binding polypeptide
or protein retains at least 85% of initial antigen-binding activity
during storage of 6 months at a temperature between RT to
40.degree. C.
14. The method according to claim 10, wherein the disease or
condition is cancer or an autoimmune disease.
15. The method according to claim 10, wherein the antigen-binding
polypeptide or protein is an angiogenesis inhibitor, an
anti-proliferative agent or a TNF inhibitor.
16. The method according to claim 12, wherein the semifluorinated
alkane is selected from F4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and
F6H10.
17. The method according to claim 12, wherein the composition is
substantially free of water.
18. The method according to claim 12, wherein the antigen-binding
polypeptide or protein is a monoclonal antibody, a polyclonal
antibody, an antibody fragment, a fusion protein comprising an
antibody fragment, an antibody-drug conjugate, or any combination
thereof.
19. The method of claim 18, wherein the semifluorinated alkane is
selected from F4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10.
20. The method of claim 12, wherein the antigen-binding polypeptide
or protein is at a concentration of at least 0.5 mg/mL.
Description
FIELD
[0001] The present invention is in the field of antibody
compositions, in particular compositions which are useful as
pharmaceutical formulations for therapeutic or diagnostic use.
BACKGROUND
[0002] Antibody-based therapies have come to the forefront in
recent years as effective treatment options for numerous diseases
such as cancer or autoimmune diseases, in conjunction with many new
developments in antibody engineering and production
technologies.
[0003] One of the major challenges associated with antibody
formulation and delivery is maintaining the stability and form of
the antibody therapeutic, in particular for long-term storage and
for transport. Antibodies, like other types of protein-based
therapeutics, are susceptible to physical and chemical instability
under stress conditions such as temperature changes from
freeze-thawing or during transport, exposure to light, oxygen or
chemical/solvents, shear stress, and pH stress.
[0004] They may undergo denaturation (e.g. loss of tertiary and/or
secondary structure) or interact to form aggregates. While antibody
aggregation can occur in both the liquid and solid state, it is
especially problematic in liquid formulations, especially at high
concentrations of antibody. Antibodies are typically
therapeutically effective at relatively high doses. High antibody
concentrations, such as to minimize dose volumes and to make
administration more patient-friendly (e.g. subcutaneous injection
instead of intravenous infusion, decreased number of injections)
are therefore generally desirable in pharmaceutical
formulations.
[0005] Aggregation, in effect, can lead to loss of active antibody
therapeutic, leading to unreliable and ineffective dosing. More
significantly, aggregates may also exhibit toxicity and trigger
undesirable or serious immunogenic responses. Aggregation resulting
in the precipitation of large particulates which impede flow is
undesirable for any kind of parenteral application.
[0006] Antibody modification and degradation via chemical pathways
such as oxidation, deamidation, isomerization, disulfide bond
formation and other irreversible crosslinking reactions may also
occur over time and lead to inactivation of the antibody, as well
as trigger aggregation. These reactions, along with aggregation,
are often accelerated at elevated temperatures; refrigeration is
consequently almost always a prerequisite. In many of these
chemical reactions, water also plays a significant role either as a
mediator or as a reactive intermediate such as in the hydrolytic
cleavage of amide bonds. The exclusion of water, such as by
lyophilization, or freeze-drying, to form a solid-state powder
formulation may thus be an effective measure towards preparing a
stable formulation of an antibody therapeutic.
[0007] Some of the formulations of marketed therapeutic antibody or
antibody derivatives/fragments are based on lyophilized powder
formulations which need to be reconstituted under sterile
conditions with aqueous media shortly prior to administration by a
trained medical or paramedical practitioner. For example,
omalizumab (e.g. Xolair.RTM., marketed by Genentech) is available
as a lyophilized powder in a single-use vial.
[0008] The reconstitution of the lyophilized antibody in sterile
aqueous media as an extra step prior to actual administration,
however, carries the risk of improper handling (e.g. shaking) or
dosing, as well as contamination. The reconstitution step itself
may trigger aggregation if the pH or temperature of the aqueous
medium is suboptimal, the time allowed for rehydration is too short
or the vial is too aggressively shaken during the dissolving step.
The propensity for waste is also higher, as failure to properly
dissolve the lyophilized antibody product within the recommended
time period usually requires for the sample to be discarded.
[0009] It should be noted that the lyophilization process step
itself may induce aggregation and/or degradation. Additional
stabilizing excipients such as saccharides or polyols are often
added to the pre-lyophilization composition, along with other
excipients such as bulking agents. The addition of other excipients
may also be required after lyophilization in order to support the
longer shelf-life of the antibody, adding to the number of
components in the final formulation.
[0010] Ready-to-use liquid formulations would generally be
preferred by the users, due to the ease of preparation for
administration. If stable, a liquid formulation is also attractive
for the pharmaceutical manufacturer due to the avoidance of
lyophilisation, which is time-consuming and costly both during drug
development and routine manufacture. Indeed, many marketed
formulations of antibodies or antibody derivatives are
aqueous-based solutions. With aqueous formulations, the pH of the
medium can have a significant impact on the stability of the
antibody in terms of promoting or reducing the likelihood of
various degradative chemical reactions. Consequently, an optimized
buffering system is always required, along with other formulation
excipients such as antioxidant free-radical scavengers, surfactants
and other anti-aggregation additives, or preservatives in order to
provide stabilization to the antibody and counteract the various
possible degradation processes that may occur during storage in an
aqueous environment over time.
[0011] Alternative formulation options to lyophilization and
aqueous solutions are also known, such as the use of non-aqueous
liquids as carrier vehicles. For example, WO2012/121754 describes
non-aqueous, high concentration suspension formulations comprising
a hydrophobic vehicle such as sesame oil, and a viscosity-reducing
agent such as ethyl oleate, and an anti-TNF.alpha. antibody. These
compositions require addition of a viscosity-reducing agent that is
fully miscible in the carrier, in order to make the oil carriers
more amenable towards injection. Generally, the parenteral use of
lipids and oils can cause pain and other undesirable side effects
at the injection site. These types of compounds may also slow down
the release of therapeutic agent, and are not ideal if rapid or
immediate bioavailable is preferred.
[0012] Perfluorinated compounds have also been described as
possible non-aqueous liquid carriers of biologically active agents
such as proteins and peptides. For example, U.S. Pat. No. 6,458,376
describes compositions proposed for ophthalmic applications (such
as topically applied eye drops) in which therapeutic/diagnostic
compounds, including oligopeptides and protein growth factors are
suspended in perfluorocarbons or fluorinated silicone oils and in
the presence of at least one surfactant. There is no mention,
however, of such compositions comprising antibodies or antibody
fragments and derivatives.
[0013] U.S. Pat. No. 6,730,328 describes thermally stable
formulations in which non-aqueous, hydrophobic, non-reactive
vehicles such as mineral oil, perfluorodecalin, methoxyflurane,
perfluorotributylamine and tetradecane are used for suspension
compositions comprising proteins, proteinaceous compounds and
nucleic acids. The formulations are proposed for parenteral,
transdermal, mucosal, oral and enteral methods of administration,
as well as their use for long-term continuous administration and
delivery via an implantable device. However, no specific example of
a suspension of an antibody or antibody fragment or derivative in
such vehicles is disclosed, nor is there any teaching with regard
to the stability of such compositions at elevated temperatures
beyond a three month time point. The actual tissue compatibility of
these types of compositions has not been demonstrated either.
[0014] WO 2011/073134 discloses solutions of ciclosporin, a cyclic
polypeptide with a molecular weight of 1202.31 in a semifluorinated
alkane, optionally in the presence of a cosolvent such as ethanol.
Whilst suspensions and emulsions are mentioned as optional
alternatives, there is no specific disclosure of such type of
composition, or any composition comprising high molecular weight
proteinaceous species in the kiloDalton range such as antibodies or
antibody fragments or derivatives.
[0015] It is therefore an object of the present invention to
introduce novel antibody compositions which overcome any of the
limitations and disadvantages associated with formulations
currently known in the art.
SUMMARY OF THE INVENTION
[0016] In a first aspect, the invention provides a novel
composition of an antigen-binding polypeptide or protein, and a
liquid vehicle which comprises a semifluorinated alkane of the
formula RFRH, wherein RF is a linear perfluorinated hydrocarbon
segment with 4 to 12 carbon atoms, and wherein RH is a linear alkyl
group with 4 to 8 carbons. The antigen-binding polypeptide or
protein is incorporated in the composition such as to form a
dispersion or suspension.
[0017] In another aspect, the antigen-binding polypeptide or
protein may be selected from a monoclonal antibody, an antibody
fragment, a polyclonal antibody, a fusion protein comprising an
antibody fragment, or an antibody-drug conjugate.
[0018] In a further aspect, the invention provides a method for the
treatment, prevention or diagnosis of a disease or condition in a
patient in need thereof, comprising the step of administering a
composition comprising an antigen-binding polypeptide or protein,
and a liquid vehicle which comprises a semifluorinated alkane of
the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon
segment with 4 to 12 carbon atoms, and wherein RH is a linear alkyl
group with 4 to 8 carbons, and wherein the antigen-binding
polypeptide or protein is incorporated in the composition such as
to form a dispersion or suspension.
[0019] In yet another embodiment, the invention provides for a
method of stabilizing an antigen-binding polypeptide or protein,
comprising the step of mixing the antigen-binding polypeptide or
protein with a liquid vehicle comprising a semifluorinated alkane
of the formula RFRH, wherein RF is a linear perfluorinated
hydrocarbon segment with 4 to 12 carbon atoms, and wherein RH is a
linear alkyl group with 4 to 8 carbons, to form a suspension or
dispersion.
BRIEF DESCRIPTION OF THE DRAWINGS
[0020] FIG. 1. Binding activities of anti-ECSCR antibody in F6H8
stored at 25.degree. C. or 40.degree. C. for 3 or 6 months compared
to initial time point of reconstitution and to control samples of
lyophilized antibody samples stored at -80.degree. C. and
never-lyophilized samples stored in PBS at 4.degree. C. (Example
1).
[0021] FIG. 2. Binding activity of bevacizumab antibody stored in
F4H5 or F6H8 between 50.degree. C. and 80.degree. C., compared to
the antibody stored in aqueous PBS buffer (Example 3).
[0022] FIG. 3. Binding activity of Fsn1006 antibody stored in F4H5
or F6H8 between 50.degree. C. and 80.degree. C., compared to the
antibody stored in aqueous PBS buffer (Example 3).
[0023] FIG. 4. Binding activity of Fsn0503 antibody stored in F4H5
or F6H8 between 50.degree. C. and 80.degree. C., compared to the
antibody stored in aqueous PBS buffer (Example 3).
DETAILED DESCRIPTION OF THE INVENTION
[0024] In a first aspect, the present invention provides
compositions comprising an antigen-binding polypeptide or protein
and a liquid vehicle which comprises a semifluorinated alkane of
the formula RFRH, wherein RF is a linear perfluorinated hydrocarbon
segment with 4 to 12 carbon atoms, and wherein RH is a linear alkyl
group with 4 to 8 carbons. Moreover, the antigen-binding
polypeptide or protein is incorporated in the composition such as
to form a dispersion or a suspension; i.e. the antigen-binding
polypeptide or protein is dispersed or suspended in the liquid
vehicle.
[0025] Semifluorinated alkanes provide a number of advantages from
the pharmaceutical perspective. They are substantially non-toxic
and are found to be well-tolerated by various types of human and
animal tissue when administered topically or parenterally. In
addition, they are chemically inert and are generally compatible
with active and inactive ingredients in pharmaceutical
formulations. They are also capable of dissolving a large range of
compounds, such as small molecule active ingredients and many
common pharmaceutically acceptable excipients, probably due to
their inherent amphiphilicity. Moreover, semifluorinated alkanes,
when acting as vehicles for compounds that are not soluble or
poorly soluble (such as antigen-binding proteins or polypeptides),
form dispersions or suspensions with very useful physical or
pharmaceutical properties, i.e. with little or no tendency to form
solid, non-dispersible sediments.
[0026] It has been found by the inventors that the presence of a
semifluorinated alkane as a liquid vehicle in a composition
comprising an antigen-binding protein or polypeptide has a
remarkable stabilizing effect on these components. In particular,
compositions comprising semifluorinated alkane as a liquid vehicle
are capable of substantially preventing or inhibiting their
aggregation and reducing chemical degradation over a substantial
period of time, at room temperature and even at higher temperatures
such as 40.degree. C., without loss of biological activity.
[0027] It has also been found that antigen-binding protein
dispersions and suspensions in semifluorinated alkanes exhibit a
remarkable degree of physical stability. The occurrence of
flotation or sedimentation takes place slowly, leaving sufficient
time for the withdrawal of a dose after gentle shaking or swirling
of the container (e.g. a vial) with the dispersion or suspension.
The antigen-binding protein particles in semifluorinated alkane
appear to largely retain their original particle size distribution,
and are readily redispersible; poorly re-dispersible aggregates do
not appear to be formed. Importantly, this provides for a higher
level of dosing accuracy in terms of precision and
reproducibility.
[0028] In contrast, suspensions or dispersions in other chemically
inert vehicles tend to be unstable, leading to formation of dense
and poorly redispersible aggregates, and making precise dosing
challenging, or in some cases, impossible, such as leading to the
clogging of fine-gauged needles typically used for subcutaneous
injections. Aggregated protein particles also present a high risk
towards triggering adverse immunogenic reactions.
[0029] Suspensions or dispersions that are unstable tend to
separate quickly by flotation of the dispersed phase, or by its
sedimentation, depending on the relative densities of the dispersed
phase and of the continuous phase. Such behaviour is usually
accompanied by rapid formation of dense and poorly re-dispersible
aggregates. Rapid flotation or sedimentation makes accurate and
reproducible dosing challenging, if not impossible. For example, if
an injectable or ophthalmic suspension settles rapidly after
shaking, and if a first dose from the full container is not
withdrawn immediately after shaking, a dose that is withdrawn may
contain a lower-than-intended number of drug particles (or if the
container is held upside down, a large-than-intended dose will be
dispensed). Later doses withdrawn from the same container will also
then contain either too high or too low of a drug-dose per volume.
Vigorous shaking of antigen-binding polypeptides and proteins in
attempt to re-disperse poorly re-dispersible aggregates may also
further trigger their further aggregation and deterioration.
[0030] Key advantages of the present invention are brought about by
the presence of a semifluorinated alkane in the composition,
functioning as a liquid vehicle. The advantageous properties of
semifluorinated alkane-based suspensions result in superior
pharmaceutical quality and performance characteristics, and also
increase the convenience of use for the patient and/or the
healthcare provider.
[0031] Semifluorinated alkanes are linear or branched alkanes some
of whose hydrogen atoms have been replaced by fluorine. In the
semifluorinated alkanes (SFAs) used in the present invention, one
linear non-fluorinated hydrocarbon segment and one linear
perfluorinated hydrocarbon segment are present. These compounds
thus follow the general formula F(CF.sub.2).sub.n(CH.sub.2).sub.mH.
According to the present invention, n is selected from the range of
4 to 12, and m is selected from the range of 4 to 8.
[0032] A nomenclature which is frequently used for semifluorinated
alkanes designates a perfluorated hydrocarbon segment as RF and a
non-fluorinated segment as RH. Alternatively, the compounds may be
referred to as FnHm and FnHm, respectively, wherein F means a
perfluorated hydrocarbon segment, H means a non-fluorinated
segment, and n and m define the number of carbon atoms of the
respective segment. For example, F3H3 is used for
perfluoropropylpropane, F(CF.sub.2).sub.3(CH.sub.2).sub.3H.
Moreover, this type of nomenclature is usually used for compounds
having linear segments. Therefore, unless otherwise indicated, it
should be assumed that F3H3 means 1-perfluoropropylpropane, rather
than 2-perfluoropropylpropane, 1-perfluoroisopropylpropane or
2-perfluoroisopropylpropane.
[0033] Preferred semifluorinated alkanes include in particular the
compounds F4H5, F4H6, F4H8, F6H4, F6H6, F6H8, and F6H10.
Particularly preferred for carrying out the invention are F4H5,
F4H6, F6H6 and F6H8. In another particularly preferred embodiment,
the composition of the invention comprises F6H8.
[0034] Optionally, the composition may comprise more than one SFA.
It may be useful to combine SFAs, for example, in order to achieve
a particular target property such as a certain density or
viscosity. If a mixture of SFAs is used, it is furthermore
preferred that the mixture comprises at least one of F4H5, F4H6,
F6H4, F6H6, F6H8, and F6H10, and in particular one of F4H5, F4H6,
F6H6 and F6H8. In another embodiment, the mixture comprises at
least two members selected from F4H5, F4H6, F6H4, F6H6, F6H8, and
F6H10, and in particular at least two members selected from F4H5,
F6H6 and F6H8.
[0035] Liquid SFAs are chemically and physiologically inert,
colourless and stable. Their typical densities range from 1.1 to
1.7 g/cm.sup.3, and their surface tension may be as low as 19 mN/m.
SFAs of the RFRH type are insoluble in water but also somewhat
amphiphilic, with increasing lipophilicity correlating with an
increasing size of the non-fluorinated segment.
[0036] Liquid SFAs of the RFRH type are being used commercially for
unfolding and reapplying a retina, for long-term tamponade as
vitreous humour substitute (H. Meinert et al., European Journal of
Ophthalmology, Vol. 10(3), pp. 189-197, 2000), and as wash-out
solutions for residual silicon oil after vitreo-retinal surgery.
Experimentally, they have also been used as blood substitutes (H.
Meinert et al., Biomaterials, Artificial Cells, and Immobilization
Biotechnology, Vol. 21(5), pp. 583-95, 1993). These applications
have established SFAs as physiologically well tolerated compounds.
On the other hand, SFAs have not been used as excipients in
approved drug products as of today.
[0037] The composition of the invention comprises an
antigen-binding polypeptide or protein. Polypeptides and proteins
in general represent polymers of amino acid units that linked to
each other by peptide bonds. Since the size boundaries that are
often used to differentiate between polypeptides and proteins are
somewhat arbitrary, the two expressions for these molecules
should--within the context of the present invention--not be
understood as mutually exclusive: A polypeptide may also be
referred to as a protein, and vice versa. Typically, the term
"polypeptide" only refers to a single polymer chain, whereas the
expression "protein" may also refer to two or more polypeptide
chains that are linked to each other by non-covalent bonds.
[0038] More specifically, and as used within the context of the
present invention, antigen-binding polypeptides or proteins refer
to full-length and whole antibodies (also known as immunoglobulins)
in their monomer, or polymeric forms and any fragments, chains,
domains or any modifications derived from a full-length antibody
capable of specifically binding to an antigen. The antigen-binding
polypeptides or proteins may belong to any of the IgG, IgA, IgD,
IgE, or IgM immunoglobulin isotypes or classes. Fusion proteins
comprising an antibody fragment capable of specifically binding to
an antigen and antibody-drug conjugates are also within the
definition of antigen-binding polypeptides or proteins as used
herein.
[0039] A full-length antibody is a Y-shaped glycoprotein comprising
of a general structure with an Fc (fragment crystallisable) domain
and a Fab (fragment antigen binding) domain. These are structurally
composed from two heavy (H) chains and two light (L) chain
polypeptide structures interlinked via disulfide bonds to form the
Y-shaped structure. Each type of chain comprises a variable region
(V) and a constant region (C); the heavy chain comprises a variable
chain region (V.sub.H) and various constant regions (e.g. C.sub.H1,
C.sub.H2, etc.) and the light chain comprises a variable chain
region (V.sub.L) and a constant region (CO. The V regions may be
further characterized into further sub-domains/regions, i.e.
framework (FR) regions comprising more conserved amino acid
residues and the hypervariable (HV) or complementarity determining
regions (CDR) which comprise of regions of increased variability in
terms of amino acid residues. The variable regions of the chains
determine the binding specificity of the antibody and form the
antigen-binding Fab domains of an antibody.
[0040] In a preferred embodiment of the invention, the compositions
comprise an antigen-binding polypeptide or protein, wherein the
antigen-binding polypeptide or protein is selected from a
monoclonal antibody, polyclonal antibody, an antibody fragment, a
fusion protein comprising an antibody fragment, an antibody-drug
conjugate, or any combination thereof.
[0041] In a particularly preferred embodiment of the invention, the
compositions comprise an antigen-binding polypeptide or protein
selected from a monoclonal antibody (mAb). A monoclonal antibody
refers to an antibody obtained from a homogenous population of
antibodies that are specific towards a single epitope or binding
site on an antigen. Monoclonal antibodies may be produced using
antibody engineering techniques known in the art, such as via
hybridoma or recombinant DNA methods.
[0042] Also within the scope of antigen-binding polypeptides and
proteins, and monoclonal antibodies are antibody fragments. As
defined herein, antibody fragments include any region, chain,
domain of an antibody, or any constructs or conjugates thereof that
can interact and bind specifically to an antigen, and may be
monovalent, bivalent, or even multivalent with respect to binding
capability. Such antibody fragments may be produced from methods
known in the art, for example, dissection (e.g. by proteolysis) of
a full-length native antibody, from protein synthesis, genetic
engineering/DNA recombinant processes, chemical cross-linking or
any combinations thereof. Antibody fragments are commonly derived
from the combination of various domains or regions featured in
variable V region of a full-length antibody.
[0043] In an embodiment of the invention, the compositions comprise
an antigen-binding polypeptide or protein selected from an antibody
fragment, wherein the antibody fragment is a fragment
antigen-binding (Fab), a single-chain variable fragment (scFv), a
single-domain antibody, a minibody, or a diabody.
[0044] Particularly preferred antibody fragments are fragment
antigen-binding domains (Fab, also referred to as Fab') or Fab
dimers comprising of two Fab fragments linked by a disulfide
linkage. Examples of Fabs are abciximab, certolizumab, digifab, and
ranibizumab. A preferred Fab is certolizumab (also known as
certolizumab pegol), which is a recombinant humanized antibody Fab'
fragment conjugated to polyethylene glycol. Certolizumab has a
molecular mass of 91 kDa and is directed against tumour necrosis
factor alpha (TNF.alpha.).
[0045] In yet another embodiment of the invention, the compositions
may comprise a single-chain variable fragment (scFv) such as those
comprising of heavy (V.sub.H) and light (V.sub.L) chain variable
domains joined by a linker or a complexed multimeric/multivalent
constructs thereof, for example, diabodies (bivalent dimer),
triabodies (trivalent trimer), or tetrabodies (tetravalent
tetramer). Multimeric antibody fragments may also be multispecific,
for example, a bispecific diabody may comprise of two fragments
each with specificity for a different antigen. Further preferred
antibody fragments include single domain antibodies (daBs) such as
those comprising a single V.sub.H or V.sub.L domain capable of
specifically binding to an antigen. Antibody fragments also within
the scope of the invention include scFv-C.sub.H dimer constructs
i.e. a minibody.
[0046] According to a further embodiment, the composition comprises
an antigen-binding polypeptide or protein with a molecular mass
selected from at least 10 KDa, at least 15 kDa, at least 35 kDa, at
least 50 kDa, at least 70 kDa, or at least 90 kDa. Also preferred
is an antigen-binding polypeptide or protein with a molecular mass
of at least 100 kDa, such as 100-150 kDa, or even higher than 150
kDa. Particularly preferred are antigen-binding polypeptides or
proteins with a molecular mass in the range of 70 kDa to 160
kDa.
[0047] In a further embodiment of the invention, the
antigen-binding polypeptide or protein may be selected from a
fusion protein comprising an antibody fragment. An antibody
fragment may be fused to another bioactive protein or polypeptide
fragment, for example, a polypeptide toxin, enzyme, cytokine,
membrane protein, etc. Examples of a fusion protein comprising an
antibody fragment include etanercept and atacicept. Etanercept is a
recombinant human protein with a molecular mass of 150 kDa,
comprising the ligand binding portion of 75 kDa tumor necrosis
factor receptor (TNFR) fused to the Fc portion of IgG1.
[0048] In another embodiment of the invention, the antigen-binding
polypeptide or protein may be selected from an antibody-drug
conjugate, wherein the antigen-binding polypeptide or protein is
covalently linked, for example via a linker or chemically
cross-linking to a small molecule drug or a radiolabelled component
such as radionuclides. Examples of antibody-drug conjugates include
gemtuzumab ozogamicin, brentuximab vedotin, .sup.90Y-labelled
ibritumomab tiuxetan, .sup.131I-labelled tositumomab,
.sup.99mTc-labelled arcitumomab. As used herein, the term
antibody-drug conjugate may also refer to an antigen-binding
polypeptide or protein that is substantially chemically modified,
for example, by PEGylation (e.g. certolizumab pegol, a PEGylated
Fab' fragment of a humanized TNF inhibitor monoclonal antibody) or
lipidation.
[0049] As understood herein, antigen-binding polypeptides and
proteins may be chimeric, humanized or human. Chimeric monoclonal
antibodies, for example, refer to hybrid monoclonal antibodies
comprising domains or regions of the heavy or light chains derived
from antibody sequences from more than one species, for example
from murine and human antibody sequences. Humanized monoclonal
antibodies refer to those that are predominantly structurally
derived from human antibody sequences, generally with a
contribution of at least 85-95% human-derived sequences, whereas
the term human refer to those are derived solely from human
germline antibody sequences. In a preferred embodiment, the
compositions comprise of an antigen-binding polypeptide or protein
selected from a monoclonal antibody, wherein the monoclonal
antibody is a chimeric, humanized, or human antibody.
[0050] In another embodiment, the composition may comprise a
polyclonal antibody, or a heterogenous mixture of antibodies
capable of recognizing more than one epitope of an antigen.
[0051] In a preferred embodiment, the antigen-binding polypeptide
or protein is a therapeutic or diagnostic compound or a vaccine. As
used herein, a therapeutic compound is a compound that is useful
for preventing a disease or condition, alleviating any symptoms of
a disease or condition, improving any disease or condition,
delaying the progress of a disease or condition or the like. A
diagnostic compound is useful for determining the state of an
organism, or for diagnosing a disease, condition, symptom, or
patient phenotype. The therapeutic compound must be administered to
the patient, whereas the diagnostic agent may be used in vivo or in
vitro, depending on the specific case. For the avoidance of doubt,
the therapeutic or diagnostic compound is incorporated within the
composition of the invention in a therapeutically or diagnostically
effective amount.
[0052] In a particularly preferred embodiment, the compositions of
the invention comprise a monoclonal antibody or antibody fragment
that is therapeutically effective or which may be administered for
the treatment of a disease or condition, such as an autoimmune
disease or inflammatory condition, a neurological disorder, or
cancer. Exemplary monoclonal antibodies or antibody fragments for
the treatment of cancer include alemtuzumab, bevacizumab,
cetuximab, gemtuzumab, ipilimumab, ibritumomab, nimotuzumab,
ofatumumab, panitumumab, rituximab, tositumomab, and trastuzumab.
Exemplary monoclonal antibodies or antibody fragments for the
treatment of autoimmune or inflammatory conditions include
adalimumab, alemtuzumab, belimumab, briakinumab, canakinumab,
eculizumab, epratuzumab, efalizumab, golimumab, infliximab,
mepolizumab, natalizumab, ofatumumab, ocrelizumab, otelixizumab,
omalizumab, reslizumab, rituximab, teplizumab, tocilizumab,
ustekinumab, and vedolizumab. Further examples of monoclonal
antibodies or antibody fragments which may be administered for the
treatment, prevention or diagnosis of a disease or condition
include basiliximab, daclizumab, denosumab, eculizumab,
palivizumab, and motavizumab.
[0053] Suspension or dispersion compositions according to the
invention may in particular comprise a cancer therapy agent
selected from an antigenbinding polypeptide or protein such as a
monoclonal antibody, polyclonal antibody, an antibody fragment, a
fusion protein comprising an antibody fragment, an antibody-drug
conjugate, or any combination thereof and a liquid vehicle
comprising a semifluorinated alkane of the formula RFRH wherein RF
is a linear perfluorinated hydrocarbon segment with 4 to 12 carbon
atoms, and wherein RH is a linear alkyl group with 4 to 8 carbon
atoms. Antigen binding polypeptides or proteins which act as
angiogenesis inhibitors or which are able to inhibit tumour cell
proliferation (anti-proliferative agent) are particularly relevant.
The antibody fragment may be a fragment antigen-binding (Fab), a
single-chain variable fragment (scFv), a single-domain antibody, a
minibody, or a diabody.
[0054] For example, a composition according to the invention may
comprise of bevacizumab and a liquid vehicle, wherein the liquid
vehicle comprises a semifluorinated alkane of the formula RFRH,
wherein RF is a linear perfluorinated hydrocarbon segment with 4 to
12 carbon atoms, and wherein RH is a linear alkyl group with 4 to 8
carbon atoms; and wherein bevacizumab is incorporated in the
composition in the form a dispersion or suspension. Antigen-binding
fragments of bevacizumab are also contemplated. Bevacizumab
(tradename Avastin.RTM.) is a humanized murine antibody that
targets VEGF-A (vascular endothelial growth factor A) and which
acts as an angiogenesis inhibitor. Compositions of the invention
comprising bevacizumab may be used for the treatment or prevention
of diseases and conditions such as colorectal, lung, breast, renal
or brain (glioblastoma) cancers, as well as eye conditions such as
age-related macular degeneration (AMD).
[0055] Further compositions may comprise the antibodies known under
the pipeline name as Fsn0503 and Fsn1006. Fsn1006 is a
dual-specific antibody that can bind to the human EGFR (epidermal
growth factor receptor) ligands amphiregulin and HB-EGF
(heparin-binding epidermal growth factor), and which can act to
inhibit cell-proliferation. Fsn1006 is a humanized IgG1/kappa
isotype. It has been demonstrated that Fsn1006 works independently
of the K-ras mutational status of the cell and therefore has
significant advantages over current EGFR targeting properties such
as cetuximab. Fsn0503 is also a humanized IgG1/kappa antibody that
targets and inhibits the proteolytic activity of human Cathepsin S.
Fsn0503 may be used in the treatment of cancer, and other
angiogenesis related diseases, in particular diseases where
Cathepsin S-mediated remodelling of the extracellular matrix is
involved.
[0056] In a yet a further preferred embodiment, suspension or
dispersion compositions according to the invention may comprise a
TNF inhibitor selected from an antigen-binding polypeptide or
protein such as an monoclonal antibody, polyclonal antibody, an
antibody fragment, a fusion protein comprising an antibody
fragment, an antibody-drug conjugate, or any combination thereof,
and a liquid vehicle comprising a semifluorinated alkane of the
formula RFRH wherein RF is a linear perfluorinated hydrocarbon
segment with 4 to 12 carbon atoms, and wherein RH is a linear alkyl
group with 4 to 8 carbon atoms. Exemplary TNF inhibitors are
infliximab, etanercerpt and certolizumab and their biosimilars.
Compositions of the invention may comprise these TNF inhibitors and
a liquid vehicle, wherein the liquid vehicle consists of
semifluorinated alkane selected from F4H5, F4H6, F4H8, F6H4, F6H6,
F6H8, and F6H10. In particular, these compositions may be used in
the therapy of autoimmune diseases affecting the gastrointestinal
system such as Crohn's disease, ulcerative colitis, or conditions
affecting the joints and skin such as rheumatoid arthritis,
psoriatic arthritis, ankylosing spondylitis and plaque
psoriasis.
[0057] In yet another embodiment, the compositions of the invention
comprise an antigen-binding polypeptide or protein at a
concentration of preferably at least 0.5 mg/mL, such as 0.5-10
mg/ml. In further preferred embodiments, the concentration is at
least 1 mg/mL, at least 5 mg/mL, at least 10 mg/mL, at least 15
mg/mL, at least 25 mg/mL or at least 35 mg/mL.
[0058] In a further aspect, the current invention presents a method
of stabilizing an antigen-binding polypeptide or protein,
comprising the step of mixing the antigen-binding polypeptide or
protein with a liquid vehicle comprising a semifluorinated alkane.
The semifluorinated alkane is of the formula RFRH, wherein RF is a
linear perfluorinated hydrocarbon segment with 4 to 12 carbon
atoms, and wherein RH is a linear alkyl group with 4 to 8 carbon
atoms. According to the method, the step of mixing the
antigen-binding polypeptide or protein with the liquid vehicle is
performed such as to form a suspension or a dispersion. Such method
of stabilizing an antigen-binding polypeptide or protein may be
useful for the preparation of a composition for use as a medicine,
such as in the treatment, prevention or diagnosis of a disease or
condition in a patient in need thereof. Optionally, such method of
stabilization may also be used for the preparation, manufacture or
synthesis of an antigen-binding polypeptide or protein.
[0059] As used herein, the term stability is defined as the
maintenance of the chemical or physical integrity and/or
bioactivity of the antigen-binding polypeptide or protein over a
period of time. Stabilizing an antigen-binding polypeptide or
protein includes the prevention or delay of degradation or
deterioration of the antigen-binding polypeptide or protein from
its biologically and/or therapeutically active form to an inactive
form. Instability may arise from events such as aggregation,
denaturation, fragmentation, or chemical modifications such as
oxidation, cross-linking, deamidation and reactions with other
components featured in the composition comprising the
antigen-binding polypeptide or protein.
[0060] The stability of the antigen-binding protein or polypeptide
in the composition may be characterized using known methods in the
art, including but not limited to, measurement of biological
activity such as antigen-binding activity with immunoassay
techniques such as ELISA, or other techniques of determining purity
or physical/chemical changes to the antigen-binding protein or
polypeptide such as size exclusion chromatography, capillary gel
electrophoresis, circular dichroism, or mass spectrometry.
Stability is determined by comparison of measurements obtained via
these types of characterization methods at an initial time point,
such as at the time of formulation or preparation of the
composition (i.e., as the case may be, the suspension or
dispersion), and those obtained at a later time point, that is,
after storage in a given environment or condition.
[0061] It has been found by the inventors that an antigen-binding
protein in a liquid vehicle comprising a semifluorinated alkane
remains stable at 25.degree. C. for at least 6 months. More
remarkably, the antigen-binding protein was comparably stable when
stored at a temperature of 40.degree. C. over the same period of
time. That is, the composition comprising the antigen-binding
protein retained effectively the same or similar antigen-binding
activity to its initial antigen-binding activity.
[0062] In a preferred embodiment, the compositions of the invention
retain at least 85% or at least 90%, such as 90-95%, or even more
than 95% of their initial antigen-binding activity during storage
of 3 months at 25.degree. C., or at room temperature, or at a
temperature between room temperature and 40.degree. C. In another
preferred embodiment, the compositions of the invention retain at
least 85% or at least 90%, such as 90-95%, or even more than 95% of
their initial antigen-binding activity, during storage of 6 months
at 25.degree. C., or at room temperature (RT), or at a temperature
between RT and 40.degree. C. In yet another embodiment, the
compositions of the invention retain at least 85% of their initial
antigen-binding activity, during storage of 6 months between RT and
40.degree. C. and a relative humidity of between 50-75%. In other
embodiments, the time period of storage may be 4-6 weeks, 6-12
weeks, or 3-6 months or 6-12 months. In further embodiments, the
humidity (RH) during storage may be at least 40% or at least 50%,
or at least 65% or at least 75%.
[0063] As mentioned, the antigen-binding polypeptide or protein is
incorporated in the composition such as to form a dispersion or
suspension. In other words, the antigen-binding polypeptide or
protein is dispersed or suspended in the liquid vehicle comprising
a semifluorinated alkane. Whether a suspension is formed upon
dispersing the antigen-binding protein in the liquid carrier
depends e.g. on the nature of the antigen-binding protein, its
concentration in the carrier, and the selected SFA(s).
[0064] As used herein, a suspension may be defined as a type of
dispersion, i.e. a system having at least one continuous (or
coherent) phase and at least one discontinuous (or inner) phase
which is dispersed in the continuous phase. In a suspension, the
dispersed phase is in the solid state. The suspensions useful for
practising the invention are liquids, at least at physiological
temperature, which means that the continuous phase is a liquid.
Typically, the suspensions are also liquid at room temperature.
Beyond suspensions, the term dispersions is understood to include
colloidal systems in which an antigen-binding protein and
polypeptide is finely dispersed in the liquid phase. In some
embodiments, the antigen-binding polypeptide or protein is also at
least partially solvated.
[0065] A stabilized suspension or dispersion of an antigen-binding
polypeptide or protein is prepared via a method comprising the step
of mixing the antigen-binding polypeptide or protein with a liquid
vehicle comprising a semifluorinated alkane. The stability of the
resulting suspension or dispersion may be characterized by the
measurement of various physical attributes including but not
limited to, for example the re-dispersibility of the suspended
particles, particle size distribution and particle size growth over
time, using such methods as those known in the art.
[0066] In one particular embodiment, the composition comprises only
the antigen-binding polypeptide or protein and one or more SFAs,
i.e. the composition consists of the antigen-binding polypeptide or
protein and one or more SFAs as defined above. In another preferred
embodiment, the composition comprising an antigen-binding
polypeptide or protein and one or more SFAs is effectively or
substantially free of water, i.e. the composition comprises no
water, except perhaps for residual amounts of water introduced via
other solid or liquid components or the antigen-binding polypeptide
or protein itself. In other cases, the suspension or dispersion
compositions comprising an antigen-binding polypeptide or protein
and a liquid vehicle comprising a semifluorinated alkane may be
water-free.
[0067] In contrast to some other suspensions or dispersions known
in prior art, the formulations of the invention require no
surfactant, or only small amounts of surfactant, for their physical
stabilisation. This is a significant advantage as surfactants have
a substantial potential for irritation and local toxicity,
especially when administered by subcutaneous or intramuscular
injection or by instillation into the eye. According to one of the
preferred embodiments, the compositions of the invention are
substantially free of surfactant. In a further embodiment, the
total amount of surfactant or surfactants, if more than one
surfactant is incorporated, is not more than about 10 wt.-%, in
particular not more than about 5 wt.-%, or preferably not more than
about 2 wt.-%, respectively. In further preferred embodiments, the
amount is not more than about 1 wt.-%, or not more than about 0.5
wt.-%, respectively. In this context, the SFAs as described herein,
although they possess some amphiphilic properties due to their
chemical structure which includes fluorinated and non-fluorinated
alkyl (or alkylene) groups characterised by different degrees of
lipophilicity, are not understood as being within the scope of
surfactants.
[0068] The surfactants which are absent or only present in small
amounts include non-ionic, cationic, anionic, and zwitterionic
surfactants as commonly used as excipients in various types of
pharmaceutical compositions, e.g. as wetting agents, emulsifiers,
dispersing agents, solubilisers and the like. Examples of
surfactants which are considered potentially useful include
tyloxapol, poloxamers such as Pluronic F68LF or Lutrol F68,
Pluronic L-G2LF and Pluronic L62D, polysorbates such as polysorbate
20 and polysorbate 80, polyoxyethylene castor oil derivatives,
sorbitan esters, polyoxyl stearates, lecithins, purified or
synthetic phospholipids, and mixtures of two or more thereof.
[0069] The compositions of the invention may optionally comprise a
non-fluorinated organic liquid, for example in order to modify the
properties of the liquid vehicle, such as the viscosity. Such other
liquid may be an oil selected from glyceride oils, liquid waxes,
and liquid paraffin, or an organic solvent exhibiting a high degree
of biocompatibility, or a mixture of more than one liquid
excipients.
[0070] Examples of potentially useful oily excipients which may be
used in combination with one or more SFA's include triglyceride
oils (i.e. soybean oil, olive oil, sesame oil, cotton seed oil,
castor oil, sweet almond oil), mineral oil (i.e. petrolatum and
liquid paraffin), medium chain triglycerides (MCT), oily fatty
acids, isopropyl myristate, oily fatty alcohols, esters of sorbitol
and fatty acids, oily sucrose esters, or any other oily substance
which is physiologically tolerated by the eye.
[0071] Examples of potentially useful organic solvents include
glycerol, propylene glycol, polyethylene glycol, and ethanol. The
concentration of the cosolvent should preferably be low relative to
that of the SFA or SFA mixture. If an organic solvent such as
ethanol is used, it is recommendable to keep it below a level of
approx. 5 wt.-%. More preferably, the content of ethanol is from
about 0.1 to about 2 wt.-%, and most preferably not more than about
1 wt.-%.
[0072] The composition may of course comprise further
pharmaceutical excipients as required or useful. Potentially useful
excipients include acids, bases, antioxidants, stabilisers,
synergists, colouring agents, thickening agents, and--if required
in a particular case--a preservative. Generally, however, the
invention provides a means of formulating non-aqueous compositions
which are microbiologically stable. This is due to the fact that
SFAs are not normally prone to microbial contamination. Hence, it
is possible to formulate preservative-free compositions to be
filled in multi-use containers. Preservative-free compositions are
better tolerated by many patients and enable lower costs of final
goods.
[0073] The liquid suspensions of the invention may be prepared by
conventional methods. In principle, the solid particles comprising
the active ingredient may be dispersed in the liquid vehicle
comprising the SFA. Alternatively, the particles may be
precipitated in situ by adding a--typically organic--solution of
the active ingredient (and, optionally, one or more solid
excipients) under controlled conditions to the SFA-based
vehicle.
[0074] The solid particles may be prepared by lyophilization or
spray-drying of a solution of the antigen-binding proteins or
particles. The solution may be aqueous or non-aqueous and may
further comprise pharmaceutical excipients as may be useful or
required.
[0075] The particle size of the dispersed phase may be also
adjusted before or after the particles are combined with the liquid
vehicle. In one of the preferred embodiments, particles of the
active ingredient are provided which already have the appropriately
selected particle size. Powders having such selected particle size
may be obtained directly from the synthesis of the respective
compound by crystal engineering, or after synthesis by conventional
grinding or milling methods using standard equipment such as a ball
mill, hammer mill, roller mill, colloidal mill, jet mill, or the
like. If the particle size is to be reduced after preparation of a
suspension, ultrasonication as well as various types of
homogenisers may be used, such as colloid mills or high pressure
homogenisers.
[0076] The current invention also provides for a method for
treating, preventing or diagnosing a disease or condition in a
patient in need thereof, comprising the step of administering to
the patient a composition, preferably in the form of a suspension
or dispersion, comprising an antigen-binding polypeptide or protein
and a liquid vehicle, wherein the liquid vehicle comprises a
semifluorinated alkane. The superior physical properties of the
suspensions according to the invention render these compositions
particularly useful for topical administration to the eye of a
patient, to the ear, nose or lung, or parenterally by injection.
For example, the compositions of the invention may be administered
to the eye of a patient by topical application or injection.
Preferred modes of injection include dermal, subcutaneous,
intramuscular, and locoregional injection. Most preferred are the
subcutaneous and intramuscular routes of administration.
EXAMPLES
Example 1
[0077] The stability of an anti-ECSCR (endothelial cell-specific
chemotaxis regulator) monoclonal antibody (clone id 13G11 1A31 A7,
a murine IgG/k antibody expressed from hybridoma cells that binds
to the endothelial marker ECSCR) in F6H8 at 25.degree. C. and at
40.degree. C. was studied over a period of 6 months.
[0078] Lyophilized 0.25 mg samples of the anti-ECSCR monoclonal
antibody (originally stored at -80.degree. C. in PBS buffer) were
reconstituted to a concentration of 1 mg/mL in F6H8. The
reconstituted samples were stored in crimped glass vials at
25.degree. C./60% RH and at 40.degree. C./75% RH. The binding
activities of these samples were determined after 3 months and 6
months of storage.
[0079] To serve as a first control, other samples of the
lyophilized antibody were stored in the lyophilized form at
-80.degree. C. These were reconstituted in PBS only prior to
analysis. A second control consisted of samples of the antibody in
PBS which were never lyophilized, but kept under refrigeration at
4.degree. C.
[0080] The binding activities of the antibody samples to ECSCR
antigen were determined by ELISA using the following protocol:
[0081] A Nunc MaxiSorp.TM. flat bottom Elisa plate was prepared by
coating with ECSCR antigen and an unrelated negative antigen. The
antigens were diluted in a coating buffer at a concentration of 1
.mu.g/mL and incubated with the plates overnight at 4.degree. C.
The plates were then blocked with 200 .mu.L of 3% blocking solution
and incubated at RT on a shaker for 1-2 hours, followed by three
washes with PBS-Tween 20 solution and blotted dry. The antibody
samples were diluted to 1 .mu.g/mL in PBS. 100 ng or 10 ng of each
diluted sample was added to a well coated with the ECSCR antigen
and a well coated with the negative control antigen. The plate was
incubated at RT for 1 h on a shaker, then washed with PBS-Tween 20
and blotted dry. The antibody aliquots were probed with
goat-anti-mouse IgG-HRP secondary antibody conjugate (Biorad,
Catalogue Nr. 170-6516) at a 1:5000 dilution in PBS. The plates
were incubated for 1 h at RT on a shaker, followed by three washes
with PBS-Tween 20 solution and blotted dry. The plate was incubated
at 37.degree. C. for 10 min with 100 .mu.L of TMB (3, 3', 5,
5'-tetramethylbenzidine), followed by addition of 50 .mu.L/well of
1M HCl. Absorbance was measured at 450 nm with a
spectrophotometer.
[0082] Comparison to the binding activities determined at the
initial time point of reconstitution and the control samples show
that the binding activity (determined from average of three
samples) of the antibody in F6H8 stored at 25.degree. C. or even at
the higher temperature of 40.degree. C. is maintained after storage
over a 6-month period (FIG. 1). The binding activity is comparable
to that observed for lyophilized anti-ECSCR monoclonal antibody
samples stored at -80.degree. C. (Control 1, FIG. 1) and for a
never-lyophilized sample stored at 4.degree. C. in PBS (Control 2,
FIG. 1.)
Example 2
[0083] The stability of bevacizumab, Fsn1006 and Fsn0503 was
studied at 25.degree. C., 50.degree. C., and 70.degree. C. over a
period of 4 weeks.
[0084] Lyophilization Protocol:
[0085] Stock solutions of each of the antibodies listed above were
obtained; Fsn1006 and Fsn0503 as solutions in PBS, bevacizumab
(tradename Avastin.RTM.) in its commercial storage buffer. An
equivalent of 80 mg of bevacizumab was transferred to >7000 MWCO
dialysis tubing and dialysed for 72 hours against 3.times.2 L
volumes of PBS. The antibody solutions were diluted to 0.5 mg/ml in
PBS and 500 .mu.l of each solution was aliquoted into individual
5-ml amber glass pharmaceutical grade vials for lyophilisation.
Lyophilisation was done over a 48 hour cycle.
[0086] Resuspension of the Lyophilized Antibodies:
[0087] The lyophilized antibodies were suspended in F4H5, F6H8, 50%
vol F4H5 in F6H8, PBS (137 mM NaCl, 2.7 mM KCl, 10 mM
Na.sub.2HPO.sub.4, 1.8 mM KH.sub.2PO.sub.4, pH=7.4) at a
concentration of 0.5 mg/mL with careful vortexing. Resuspension of
the lyophilized antibodies in PBS forms solutions, while
suspensions were formed with the semifluorinated alkanes. The
resulting suspensions and solutions were stored in amber glass
vials at 25.degree. C. and 50.degree. C. for 4 weeks (28 days)
After day 23, the 50.degree. C. samples were subjected to
70.degree. C. conditions.
[0088] The binding activities of the antibody samples were
determined by ELISA testing at t=0 (immediately after
resuspension), t=2 weeks and t=4 weeks. As a control, lyophilized
antibodies samples which were never re-suspended (stored at
-80.degree. C.) and solutions of the antibodies in PBS which were
never lyophilized (stored at 4.degree. C.) were used.
[0089] ELISA Protocol:
[0090] For the ELISA testing of each antibody, a Nunc Maxisorp 96
well plate was coated with the target antigen by adding 100 ng/well
of the appropriate antigen in 100 .mu.l 0.2 M carbonate buffer, pH
9.5 and incubated for 1 hour at 37.degree. C. After washing with
PBS containing 0.1% tween 20 (PBS-T), the plate was blocked by
adding 200 .mu.l 4% milk powder in PBS and incubating at room
temperature for 2 hours on a shaker. After further washing, the
antibody samples were applied. 1 ml of .times.0.5 PBS was added to
each non-aqueous vial. The antibody was extracted into the solution
by gentle rocking for 5 minutes. 1 .mu.l of the aqueous layer was
then transferred to a vial containing 999 .mu.l PBS (to give a
nominal value of 50 ng/well). 100 .mu.l of each sample vial was
then plated in 6 replicates of 100 .mu.l. The plates were incubated
overnight at 4.degree. C. After washing, the secondary antibody was
applied (goat anti-human HRP conjugate) at a 1:60,000 dilution in
PBS; 100 .mu.l was added to each well and the plate was incubated
for 1 hour at room temperature with shaking. The plates were then
washed with 3 volumes of PBS-T followed by 2 volumes of PBS. 100
.mu.l of TMB solution was then applied and the plate incubated for
10 minutes at 37.degree. C. The reaction was stopped by the
addition of 50 .mu.l 1M HCl. Absorbance at .lamda.=450 nm was read
for each well.
TABLE-US-00001 TABLE 1 Bevacizumab Activity (Mean OD .+-. 2SD at
450 nm) 50% vol. Week F4H5 F6H8 F4H5 in F6H8 PBS 25.degree. C. 0
3.15 .+-. 0.19 3.26 .+-. 0.11 3.12 .+-. 0.12 3.04 .+-. 0.03 2 3.23
.+-. 0.05 2.34 .+-. 0.09 3.05 .+-. 0.10 2.12 .+-. 0.07 4 2.16 .+-.
0.36 2.23 .+-. 0.46 2.03 .+-. 0.56 0.30 .+-. 0.54 50.degree. C.
(70.degree. C. last 5 days) 0 3.16 .+-. 0.01 3.26 .+-. 0.11 3.14
.+-. 0.05 3.00 .+-. 0.05 2 3.27 .+-. 0.09 3.19 .+-. 0.10 3.06 .+-.
0.19 3.16 .+-. 0.08 4 1.63 .+-. 0.62 1.69 .+-. 0.34 1.64 .+-. 0.54
0.05 .+-. 0.003
TABLE-US-00002 TABLE 2 Fsn1006 Activity (Mean OD .+-. 2SD at 450
nm) 50% vol. Week F4H5 F6H8 F4H5 in F6H8 PBS 25.degree. C. 0 2.57
.+-. 0.20 2.47 .+-. 0.94 2.12 .+-. 0.21 2.63 .+-. 0.21 2 3.27 .+-.
0.13 3.32 .+-. 0.15 3.26 .+-. 0.10 2.99 .+-. 0.10 4 2.54 .+-. 0.30
2.43 .+-. 0.45 2.45 .+-. 0.38 0.62 .+-. 1.26 50.degree. C.
(70.degree. C. last 5 days) 0 2.56 .+-. 0.27 2.47 .+-. 0.94 2.26
.+-. 0.20 2.56 .+-. 0.24 2 3.32 .+-. 0.04 3.28 .+-. 0.04 2.81 .+-.
0.04 2.79 .+-. 0.13 4 2.01 .+-. 0.40 1.97 .+-. 0.24 1.80 .+-. 0.91
0.05 .+-. 0.01
TABLE-US-00003 TABLE 3 Fsn0503 Activity (Mean OD .+-. 2SD at 450
nm) 50% vol. Week F4H5 F6H8 F4H5 in F6H8 PBS 25.degree. C. 0 3.07
.+-. 0.10 3.06 .+-. 0.08 2.98 .+-. 0.22 3.11 .+-. 0.16 2 3.22 .+-.
0.23 3.22 .+-. 0.09 3.20 .+-. 0.08 2.01 .+-. 1.23 4 2.70 .+-. 0.19
2.43 .+-. 0.19 2.87 .+-. 0.24 0.72 .+-. 1.98 50.degree. C.
(70.degree. C. last 5 days) 0 3.09 .+-. 0.08 3.06 .+-. 0.08 3.07
.+-. 0.08 3.11 .+-. 0.07 2 3.26 .+-. 0.10 3.22 .+-. 0.12 3.18 .+-.
0.14 3.10 .+-. 0.21 4 2.64 .+-. 0.22 2.19 .+-. 0.18 2.73 .+-. 0.19
0.65 .+-. 1.91
[0091] The activity of bevacizumab, Fsn1006 and Fsn0503 stored as
suspensions in F4H5, F6H8 and 50% vol F4H5 in F6H8 was
substantially consistent during a 4-week period at both 25.degree.
C. and at 50.degree. C., including the increase to a storage
temperature of 70.degree. C. for the 50.degree. C. sample during
the last five days of the period. In contrast the binding activity
demonstrated by the solutions of these antibodies in PBS buffer
deteriorated significantly by the end of the four-week period
regardless of storage temperature.
Example 3
[0092] The binding activity of bevacizumab, Fsn1006, and Fsn0503
stored in SFA and PBS buffer at temperatures between 50.degree. C.
and 80.degree. C. was examined.
[0093] The lyophilized antibodies bevacizumab, Fsn1006, and Fsn0503
(as prepared above in Example 2) were re-suspended in F4H5, F6H8,
and PBS at a concentration of 0.5 mg/ml with careful vortexing.
Re-suspension of these lyophilized antibodies in PBS yields
solutions, while suspensions were formed with the SFAs.
[0094] The suspension/solution samples were held at temperatures of
50.degree. C., 55.degree. C., 60.degree. C., 65.degree. C.,
70.degree. C., 75.degree. C., and 80.degree. C. respectively for a
period of 2 h and then cooled to 10.degree. C. prior to PBS buffer
extraction and activity assay using the ELISA method as described
above in Example 2. Each experiment was performed in triplicate.
Samples heated at 40.degree. C. were used as a control.
[0095] It was found that the bevacizumab (FIG. 2), Fsn1006 (FIG. 3)
and Fsn0503 (FIG. 4) suspensions in F4H5 and F6H8 demonstrated
significant stability towards thermal denaturation compared to the
samples in aqueous buffer. Binding activity remained fairly
constant between 50.degree. C. and 80.degree. C. for the antibodies
formulated in these semifluorinated alkanes. In contrast, the
activity of these antibodies stored in aqueous PBS buffer
deteriorated sharply at temperatures above 60.degree. C.
Example 4
[0096] The stability of a composition comprising an antigen-binding
polypeptide or protein selected from infliximab (a chimeric
monoclonal antibody with human constant and murine variable
regions, and a molecular mass of 149 kDa), the fusion protein
etanercept (a recombinant human protein with a molecular mass of
150 kDa comprising the ligand binding portion of the 75 kDa TNFR
(tumor necrosis factor receptor) fused to Fc portion of IgG1), and
certolizumab (an antibody fragment with a molecular mass of 91 kDa,
comprising a recombinant Fab' conjugated to an approximately 40 kDa
polyethylene glycol) or biosimilars thereof and a liquid vehicle
comprising a semifluorinated alkane of formula RFRH wherein RF is a
linear perfluorinated hydrocarbon segment with 4 to 12 carbon
atoms, and wherein RH is a linear alkyl group with 4 to 8 carbon
(for example F4H5 and F6H8) is studied and compared to formulations
in different media.
[0097] The antibody is lyophilized and re-suspended in a
semifluorinated alkane. Comparative suspensions/solutions are also
prepared in other media. The following stability tests are
performed on these formulations: [0098] Storage stability under ICH
conditions (25 and 40.degree. C.). [0099] Temperature stability
outside of ICH conditions, i.e. thermal denaturing
[0100] These are carried out in analogy to the methods described in
examples 1-3. Analytical methods suitable for assaying the
stability of these antigen-binding polypeptides are performed.
Assays to monitor the activity and potency of the antibody, as well
as to monitor aggregation levels throughout the course of the
stability experiments listed above include techniques such as ELISA
in similarity to the protocol as described above in Examples
1-3.
[0101] The analytical methods are carried out with samples obtained
directly from the antibody suspensions or depending on the
analytical technique, on samples which are aqueous buffer
extractions.
[0102] It is expected that the compositions comprising the antigen
binding polypeptide or proteins will exhibit improved stability and
a decreased propensity towards aggregate formation.
* * * * *