U.S. patent application number 13/393636 was filed with the patent office on 2012-09-27 for stable formulations of polypeptides and uses thereof.
This patent application is currently assigned to Ablynx N.V.. Invention is credited to Ann Brige, Veronique De Brabandere.
Application Number | 20120244158 13/393636 |
Document ID | / |
Family ID | 42797362 |
Filed Date | 2012-09-27 |
United States Patent
Application |
20120244158 |
Kind Code |
A1 |
Brige; Ann ; et al. |
September 27, 2012 |
STABLE FORMULATIONS OF POLYPEPTIDES AND USES THEREOF
Abstract
Formulations are provided that contain single variable domains
with a good solubility and good stability under different storage,
transportation and stress conditions. The formulations are useful
as pharmaceutical formulation. The formulation comprises an aqueous
carrier with a pH of 5.5 to 8.0, a buffer selected from the group
consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,
succinate pH 6.0-6.5 and acetate pH 5.5-6.0; an excipient; and/or a
surfactant selected from Tween 80, Tween 20 and poloxamers. The
formulation is further characterized that it has an inorganic salt
concentration of 150 mM or lower. The invention further relates to
containers and pharmaceutical units comprising such formuSations
and to methods for preparing and prophylactic and therapeutic uses
of the formulations and pharmaceutical units of the invention.
Inventors: |
Brige; Ann; (Ertvelde,
BE) ; De Brabandere; Veronique; (Gent, BE) |
Assignee: |
Ablynx N.V.
Zwijnaarde
BE
|
Family ID: |
42797362 |
Appl. No.: |
13/393636 |
Filed: |
September 3, 2010 |
PCT Filed: |
September 3, 2010 |
PCT NO: |
PCT/EP2010/062975 |
371 Date: |
April 24, 2012 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
61275816 |
Sep 3, 2009 |
|
|
|
61284502 |
Dec 18, 2009 |
|
|
|
Current U.S.
Class: |
424/135.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
A61K 39/39591 20130101; A61K 47/26 20130101; A61P 19/10 20180101;
A61P 19/08 20180101; A61K 47/14 20130101; A61K 47/02 20130101 |
Class at
Publication: |
424/135.1 |
International
Class: |
A61K 39/395 20060101
A61K039/395 |
Claims
1. A formulation comprising an aqueous carrier having a pH of 5.5
to 8.0 and a polypeptide comprising one or more single variable
domains at a concentration of 1 mg/mL to 200 mg/mL, said
formulation being formulated for administration to a human subject
and said formulation further comprising one or more components
selected from: a) A buffer at a concentration of 10 mM to 100 mM
selected from the group consisting of histidine pH 6.0-6.5, hepes
pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0; b) An excipient at a concentration of 1% to 20%; c) A
surfactant at a concentration of 0.001% to 1% selected from Tween
80, Tween 20 or a poloxamer; wherein said formulation has an
inorganic salt concentration of 150 mM or lower.
2. The formulation of claim 1, that does not contain any inorganic
salt.
3. The formulation of claim 1, wherein the concentration of
polypeptide is about 1 to 200 mg/ml or more, preferably about 5 to
100 mg/mL or more, more preferably about 5 to 50 mg/mL or more,
most preferably about 5 to 30 mg/mL or more, such as around 5
mg/mL, around 10 mg/mL, around 20 mg/mL, around 30 mg/mL, around 40
mg/mL, around 50 mg/mL, around 60 mg/mL, around 70 mg/mL, around 80
mg/mL, around 90 mg/mL, around 100 mg/mL, around 150 mg/mL or even
more.
4. The formulation of claim 1, wherein the polypeptide comprises
two or more single variable domains, such as two or three.
5. The formulation of claim 4, wherein the polypeptide is selected
from one of SEQ ID NO's: 1 to 6.
6. The formulation of claim 1, wherein the polypeptide has a
solubility of at least 20 mg/mL, preferably 50 mg/mL or more, more
preferably 90 mg/mL or more or 120 mg/mL or more, most preferably
150 mg/mL or more, or even 200 mg/mL or more, as determined by the
PEG exclusion method or by a concentration experiment; the
polypeptide has a melting temperature of at least 59.degree. C. or
more, preferably at least 60.degree. C. or more, more preferably at
least 61.degree. C. or more or at least 62.degree. C. or more, most
preferably at least 63.degree. C. or more as measured by the
thermal shift assay (TSA) and/or differential scanning calorimetry
(DSC); no particulates are present as measured by OD320/OD280
and/or elastic light scattering; less than 10% of the polypeptide
forms pyroglutamate at the N-terminal glutamic acid during storage
at a temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more), the % of pyroglutamate as
measured by RP-HPLC; less than 10% of the polypeptide forms dimers
during storage at a temperature of 37.+-.5.degree. C. up to at
least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more), the %
of dimers as measured by SE-HPLC; at least 80% of the polypeptide
retains its binding activity to at least one of its targets after
storage at 37.+-.5.degree. C. up to at least 2 weeks (preferably at
least 3 weeks, at least 5 weeks, at least 2 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more) compared
to the binding activity prior to storage, said binding activity as
measured by ELISA and/or Biacore; and/or the polypeptide is stable
during mechanical stress.
7. The formulation of claim 1, wherein the pH is between 6.0 and
8.0, preferably between 6.2 and 7.5, more preferably between 6.5
and 7.5 or 6.5 and 7.0, such as 6.5 or 7.0.
8. The formulation of claim 1, wherein the buffer is a histidine
buffer.
9. The formulation of claim 8, wherein the buffer is a histidine pH
6.5 or histidine pH 6.0 buffer.
10. The formulation of claim 8, wherein the histidine buffer has a
concentration of 10 to 50 mM, more preferably 10 to 20 mM, such as
about 10 mM or 15 mM.
11. The formulation of claim 1, wherein the excipient is a
saccharide, a non-reducing sugar and/or polyol.
12. The formulation of claim 11, wherein the excipient is selected
from the group consisting of mannitol, trehalose, sorbitol and
sucrose.
13. The formulation of claim 11, wherein the excipient has a
concentration of 2.5% to 15%, more preferably 5% to 10%, such as
about 5%, 7.5%, 8% and 10%.
14. The formulation of claim 1, wherein the surfactant is Tween
80.
15. The formulation of claim 14, wherein the surfactant has a
concentration of 0.01% to 0.1%, preferably 0.01% to 0.05%, such as
about 0.01% or 0.005%.
16. The formulation of claim 1, comprising: a) A histidine pH 6.5
or pH 6.0 buffer at a concentration of 10 mM to 100 mM; b) Sucrose
at a concentration of 1% to 20%; and c) Tween 80 at a concentration
of 0.001% to 1%.
17. The formulation of claim 16, comprising: a) 15 mM histidine pH
6.5; b) 8% sucrose; and c) 0.01% Tween 80.
18. The formulation of claim 16, comprising: a) 10 mM histidine pH
6.0; b) 10% sucrose; and c) 0.005% Tween 80.
19. The formulation of claim 17, comprising: a) 15 mM histidine pH
6.5; b) 8% sucrose; c) 0.01% Tween 80; and d) A polypeptide
selected from SEQ ID NO's: 1 to 6.
20. The formulation of claim 18, comprising: a) 10 mM histidine pH
6.0; b) 10% sucrose; c) 0.005% Tween 80; and d) A polypeptide
selected from SEQ ID NO's: 1 to 6.
21. A method for the preparation of a formulation of claim 1, at
least comprising the step of concentrating the polypeptide and
exchanging it with the selected buffer and/or excipient.
22. A sealed container containing a formulation according to claim
1.
23. A pharmaceutical unit dosage form suitable for parenteral
administration to a human, comprising a formulation according to
claim 1 in a suitable container.
24. A kit comprising one or more of the sealed containers according
to claim 22 and instructions for use of the formulation.
25. The formulation of claim 1, for use in therapy.
26. Method for prevention and/or treatment of one or more diseases
and/or disorders, comprising administering to a subject in need
thereof a formulation according to claim 1.
27. Method of claim 26, wherein the disease is a disease and/or
disorder associated with aberrant expression and/or activity of
RANKL, disease and/or disorder associated with overexpression of
IL-6, or disease and disorder associated with heterodimeric
cytokines and their receptors.
28. A kit comprising one or more of the pharmaceutical unit dosage
forms according to claim 23, and instructions for use of the
formulation.
Description
FIELD OF THE INVENTION
[0001] The present invention relates to formulations of single
variable domains. More specifically the present invention provides
formulations that contain single variable domains with a good
solubility and good stability under different storage and stress
conditions. The formulations of the invention are suitable for
administration to human subjects.
[0002] The invention further relates to containers and
pharmaceutical units comprising such formulations and to
prophylactic and therapeutic uses of the formulations and
pharmaceutical units of the invention.
[0003] Other aspects, embodiments, advantages and applications of
the invention will become clear from the further description
herein.
BACKGROUND ART
[0004] Nanobodies (as further described herein) are characterized
by formation of the antigen binding site by a single variable
domain, which does not require interaction with a further domain
(e.g. in the form of VH/VL interaction) for antigen recognition.
Nanobodies have been described against a wide range of different
targets (WO 04/062551, WO 05/044858, WO 06/040153, WO 06/122825, WO
07/104,529, WO 08/020,079, WO 08/074,839, WO 08/071,447, WO
08/074,840, WO 08/074,867, WO 08/077,945, WO 08/101,985, WO
08/142,164, WO 09/068,625, WO 08/142,165, WO 09/068,627) which
could be ideal candidates for drug development. Nanobodies against
IL-6R that can inhibit the IL-6/IL-6R interaction are described in
WO 08/020,079. Nanobodies against the p19 subunit of IL-23 that
block the interaction of IL-23 with its receptor have been
described in WO 09/068,627. Nanobodies against RANKL that can
inhibit osteoclast formation are described in WO 08/142,164. The
OPG/RANKL/RANK system has recently been discovered as pivotal
regulatory factors in the pathogenesis of bone diseases and
disorders like e.g. osteoporosis.
[0005] Proteins, such as therapeutic antibodies and Nanobodies, are
often transported and/or stored for later use. It is important
therefore that such proteins preserve the stability and biological
activity of the protein under various conditions such as different
temperature regimens and mechanical stress.
[0006] Certain prior liquid antibody preparations have shown short
shelf lives and loss of biological activity of the antibodies
resulting from chemical and/or physical instabilities during the
transportation and storage. Chemical instability may be caused by
deamidation, racemization, hydrolysis, oxidation, beta elimination
or disulfide exchange, and physical instability may be caused by
antibody denaturation, aggregation, precipitation or adsorption.
Among those, aggregation, deamidation and oxidation are known to be
the most common causes of the antibody degradation (Cleland et al.,
1993, Critical Reviews in Therapeutic Drug Carrier Systems 10:
307-377). Little is known about drug formulation components that
provide stable liquid formulations of Nanobodies.
[0007] There exists a need for stable liquid formulations of
Nanobodies which show a good solubility of the Nanobody and which
exhibit increases stability, low to undetectable levels of
aggregation, low to undetectable levels of Nanobody degradation,
and very little to no loss of biological activity of the Nanobody,
even under different transportation and storage conditions.
SUMMARY OF THE INVENTION
[0008] The present invention provides improved formulations (also
referred to as "formulation(s) of the invention") of polypeptides
comprising one or more single variable domains that show good
solubility of the single variable domains and retain increased
stability of the single variable domains under a variety of
different transportation, storage and in-use conditions. The
present invention is based on the finding that the presence in the
formulation of certain buffers, certain excipients and/or certain
surfactants may increase the solubility, the melting temperature
and/or the stability of the single variable domains present in the
formulation.
[0009] The present invention provides solubility data for
formulations with polypeptides comprising one or more single
variable domains (also referred to as "polypeptide(s) of the
invention") up to 150 mg/mL and higher. The invention further shows
that such formulations can be transported, manipulated through
various administration devices and retain activity, purity and
potency under different stress conditions: mechanical stress
conditions; storage of the formulation at various stress conditions
such as different freeze/thaw cycles, at 2-8.degree. C., at
25.+-.5.degree. C. and at elevated temperature.
[0010] The formulation of the present invention comprises an
aqueous carrier with a pH of 5.5 to 8.0 and a polypeptide
comprising one or more single variable domains at a concentration
of 1 mg/ml to 200 mg/ml, said formulation being formulated for
administration to a human subject and said formulation further
comprising one or more components selected from: [0011] a) A buffer
at a concentration of 10 mM to 100 mM selected from the group
consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,
succinate pH 6.0-6.5 and acetate pH 5.5-6.0; [0012] b) An excipient
at a concentration of 1% to 20% (w:v); [0013] c) A surfactant at a
concentration of 0.001% to 1% selected from Tween 80, Tween 20 and
poloxamers; wherein said formulation has an inorganic salt
concentration of 150 mM or lower.
[0014] In a preferred aspect, the formulation of the invention
comprises at least two of the above components, such as e.g. at
least the components in a) and b), at least the components in a)
and c) or at least the components in b) and c). Preferably, the
formulation of the invention comprises the components in a), b) and
c).
[0015] The present inventors observed that formulations that have
150 mM or less of inorganic salt show a much better stability of
the single variable domains contained in the formulation. Inorganic
salts frequently used in pharmaceutical formulation are NaCl and
KCl. The single variable domains present in formulations containing
150 mM or less of inorganic salt have shown increased stability
(e.g. less tendency to form aggregates, dimers and/or
pyroglutamate, or to loose potency) at different stress storage
conditions (such as e.g. during storage at a temperature of
37.+-.5.degree. C. up to at least 2 weeks (preferably at least 3
weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more)), an improved melting temperature and/or an
increased solubility. Preferably, the formulation contains 100 mM
or less of inorganic salt, more preferably even 50 mM or less of
inorganic salt. Most preferably the formulation does not contain
any inorganic salt.
[0016] The polypeptide (also referred to as "polypeptide of the
invention") comprising one or more single variable domains for use
in the formulation of the invention may be therapeutic or
prophylactic, and may be useful in the prevention, treatment and/or
management of one or more diseases and/or disorders. In one
specific aspect, the polypeptide has at least two single variable
domains. In another specific aspect, the polypeptide has at least
three single variable domains. Preferred polypeptides of the
invention and single variable domains used in the polypeptide of
the invention are described in WO 08/142,164, WO 08/020,079 and WO
09/068,627. Particularly preferred polypeptides of the invention
may be selected from SEQ ID NO's: 1 to 6.
[0017] The polypeptide of the invention may be present in the
formulation of the present invention at a concentration of about 1
to 200 mg/mL or more, preferably about 5 to 100 mg/mL or more, more
preferably about 5 to 50 mg/mL or more, most preferably about 5 to
30 mg/mL or more, such as around 5 mg/mL, around 10 mg/mL, around
20 mg/mL, around 30 mg/mL, around 40 mg/mL, around 50 mg/mL, around
60 mg/mL, around 70 mg/mL, around 80 mg/mL, around 90 mg/mL, around
100 mg/mL, around 150 mg/mL or even more.
[0018] In an aspect of the invention, the formulation is
homogeneous. In another aspect, the formulation of the invention is
sterile. In addition to the polypeptide of the invention, the
formulation of the present invention comprises at least an aqueous
carrier (e.g. distilled water, MilliQ water or WFI) and a
buffer.
[0019] The pH of the formulation of the invention should be in the
range of 5.5 to 8.0, preferably the pH is around 6.0 to 7.5, more
preferably around 6.2 to 7.5 or around 6.2 to 7.0. Most preferably
the pH is in the range of 6.5 to 7.0, such as e.g. pH 6.5. These pH
ranges have shown to provide an increased melting temperature to
the polypeptides present in the formulation of the invention.
[0020] Preferred buffers for use in the formulation of the
invention are hepes pH 7.0-8.0, histidine pH 6.0-6.5, MES pH 6.0,
succinate pH 6.0-6.5 or acetate pH 5.5-6.0, preferably hepes pH 7.0
or histidine pH 6.0-6.5, most preferably histidine pH 6.0, 6.2 or
6.5. Formulations comprising one of these buffers have shown a very
good solubility (as defined herein) of the polypeptides of the
invention, an improved melting temperature of the polypeptides
present in the formulation and increased stability (e.g. less
tendency to form aggregates, dimers and/or pyroglutamate, or to
lose potency) at different stress storage conditions (such as e.g.
during storage at a temperature of 37.+-.5.degree. C. up to at
least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more)). The
buffer is preferably at a concentration of about 10 to 20 mM, such
as 10 mM or 15 mM. In a specific aspect, the formulation of the
invention comprises a histidine buffer pH 6.5 at a concentration of
15 mM or a histidine buffer pH 6.0 at a concentration of 10 mM.
[0021] Accordingly, the present invention provides stable
formulations of polypeptides comprising one or more single variable
domains, said formulations comprising an aqueous carrier, the
polypeptide at a concentration from about 1 to 200 mg/mL or more,
preferably about 5 to 100 mg/mL or more, more preferably about 5 to
50 mg/mL or more, most preferably about 5 to 30 mg/mL or more, such
as around 5 mg/mL, around 10 mg/mL, around 20 mg/mL, around 30
mg/mL, around 40 mg/mL, around 50 mg/mL, around 60 mg/mL, around 70
mg/mL, around 80 mg/mL, around 90 mg/mL, around 100 mg/mL, around
150 mg/mL or even more, and a buffer such as a histidine buffer
with a pH ranging from 6.0 to 7.0 at a concentration of about 10 to
20 mM.
[0022] Preferably the formulation of the invention is isotonic or
slightly hypotonic and/or has an osmolality of about 290.+-.60
mOsm/kg, such as about 240 or higher, 250 or higher or 260 or
higher. Isotonicity of the formulation can be further adjusted by
the addition of one or more excipients and/or tonifiers.
[0023] Preferred excipients/tonifiers for use in the formulation of
the present invention are saccharides and/or polyols. Accordingly,
in another aspect, the formulation of the invention comprises a
saccharide and/or polyol. Formulations comprising one or more
saccharides and/or polyols have shown increased stability (e.g.
less tendency to form aggregates, dimers and/or pyroglutamate, or
to loose potency) at different stress storage conditions (such as
e.g. during storage at a temperature of 37.+-.5.degree. C. up to at
least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more) and
during mechanical stress conditions) and/or an improved melting
temperature of the polypeptides present in the formulation. In a
specific aspect of the invention, the excipient present in the
formulation of the invention is a non-reducing sugar. In another
specific aspect, the excipient present in the formulation of the
invention is a disaccharide. In another specific aspect, the
excipient present in the formulation of the invention is selected
from sucrose, trehalose, sorbitol and mannitol. The saccharide
and/or polyol is preferably present in the formulation of the
invention at a concentration of about 1% to 20%, preferably about
2.5% to 15%, more preferably about 5% to 10%, such as around 5%,
around 7.5%, around 8% or around 10%.
[0024] Accordingly, the present invention provides stable
formulations of polypeptides comprising one or more single variable
domains, said formulations comprising an aqueous carrier, the
polypeptide at a concentration from about 1 to 200 mg/mL or more,
preferably about 5 to 100 mg/mL or more, more preferably about 5 to
50 mg/mL or more, most preferably about 5 to 30 mg/mL or more, such
as around 5 mg/mL, around 10 mg/mL, around 20 mg/mL, around 30
mg/mL, around 40 mg/mL, around 50 mg/mL, around 60 mg/mL, around 70
mg/mL, around 80 mg/mL, around 90 mg/mL, around 100 mg/mL, around
150 mg/mL or even more, and a saccharide and/or polyol at a
concentration of about 1% to 20%, preferably about 2.5% to 15%,
more preferably about 5% to 10%, such as around 5%, around 7.5%,
around 8% or around 10%.
[0025] In another specific aspect, the formulation of the invention
may comprise one or more surfactants (e.g., Tween 20, Tween 80 or a
poloxamer). Formulations comprising a surfactant have shown a very
good solubility (as defined herein) of the polypeptides of the
invention and/or increased stability under mechanical stress. The
surfactant may be present at a concentration in the range of about
0.001% to 1% (preferably between about 0.001% to 0.1%, or about
0.01% to 0.1% such as around 0.001%, around 0.005%, around 0.01%,
around 0.02%, around 0.05%, around 0.08%, around 0.1%, around 0.5%,
or around 1% of the formulation, preferably around 0.01%).
[0026] Accordingly, the present invention provides stable
formulations of polypeptides comprising one or more single variable
domains, said formulations comprising an aqueous carrier, the
polypeptide at a concentration from about 1 to 200 mg/mL or more,
preferably about 5 to 100 mg/mL or more, more preferably about 5 to
50 mg/mL or more, most preferably about 5 to 30 mg/mL or more, such
as around 5 mg/mL, around 10 mg/mL, around 20 mg/mL, around 30
mg/mL, around 40 mg/mL, around 50 mg/mL, around 60 mg/mL, around 70
mg/mL, around 80 mg/mL, around 90 mg/mL, around 100 mg/mL, around
150 mg/mL or even more, and a surfactant (e.g., Tween 20, Tween 80
or a poloxamer) at a concentration in the range of about 0.001% to
1% (preferably between about 0.001% to 0.1%, or about 0.01% to 0.1%
such as around 0.001%, around 0.005%, around 0.01%, around 0.02%,
around 0.05%, around 0.08%, around 0.1%, around 0.5%, or around 1%
of the formulation, preferably around 0.01%).
[0027] A preferred formulation of the invention may comprise:
[0028] a) A histidine pH 6.5 buffer at a concentration of 10 mM to
100 mM, such as 10 mM to 20 mM;
[0029] b) Sucrose at a concentration of 1% to 10%; and
[0030] c) Tween 80 at a concentration of 0.001% to 1%.
[0031] Another preferred formulation of the invention may
comprise:
[0032] a) A histidine pH 6.5 buffer at a concentration of 15
mM;
[0033] b) Sucrose at a concentration of 8%; and
[0034] c) Tween 80 at a concentration of 0.01%.
[0035] Another preferred formulation of the invention may
comprise:
[0036] a) A histidine pH 6.0 buffer at a concentration of 10 mM to
100 mM, such as 10 mM to 20 mM;
[0037] b) Sucrose at a concentration of 1% to 10%; and
[0038] c) Tween 80 at a concentration of 0.001% to 1%.
[0039] Another preferred formulation of the invention may
comprise:
[0040] a) A histidine pH 6.0 buffer at a concentration of 10
mM;
[0041] b) Sucrose at a concentration of 10%; and
[0042] c) Tween 80 at a concentration of 0.005%.
[0043] The present invention provides formulations of a polypeptide
comprising one or more single variable domains which exhibit high
solubility of the polypeptide, little to no aggregation of the
polypeptide and high stability during long periods of storage.
[0044] In one aspect, the components present in the formulations of
the invention have been selected such that the polypeptides of the
invention have a solubility of at least 20 mg/mL, at least 50
mg/mL, preferably at least 90 mg/mL, at least 120 mg/mL, at least
150 mg/mL or even 200 mg/mL or more.
[0045] In another aspect, the components present in the
formulations of the invention have been selected such that the
polypeptide present in the formulation of the invention has a
melting temperature of at least 59.degree. C. or more (such as
59.5.degree. C. or more), preferably at least 60.degree. C. or more
(such as 60.5.degree. C. or more), more preferably at least
61.degree. C. or more (such as 61.5.degree. C. or more) or at least
62.degree. C. or more (such as 62.5.degree. C. or more), most
preferably at least 63.degree. C. or more (such as 63.5.degree. C.
or more) as measured by the thermal shift assay (TSA) and/or
differential scanning calorimetry (DSC).
[0046] In yet another aspect, the formulation of the present
invention exhibits stability under various stress conditions such
as: [0047] multiple (up to 10) freeze/thaw cycles; [0048] storage
at a temperature of 2-8.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more); [0049] storage at a
temperature of 25.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 15 year or even 2 years or more); [0050] storage at a
temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more); and/or [0051] mechanical
stress.
[0052] Mechanical stress as used in the present invention can be
any form of external force applied on the formulation that may
affect the stability of the polypeptide present in the formulation.
Without being limiting, the mechanical stress applied to the
solution can be shear stress, stir stress, shake stress, rotation
stress, etc. Preferably the formulation of the invention is stable
under one or more of the following forms of mechanical stress:
[0053] shaking the formulation during 10 s to 1 min; [0054] pushing
the formulation through a needle (250, preferably 260, more
preferably 270, even more preferably 280, most preferably 29 G or
more) with a syringe (the syringe used can be any commercially
available syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 ml, 10
mL up to 50 mL syringe); [0055] rotating for two days at 10 rpm;
and/or [0056] stirring for 1 hour at room temperature and/or 4-48
hours (such as 4-8 hours, 12 hours, 24 hours or even 48 hours) at
4.degree. C. at at least 10 rpm (such as 50 rpm, 100 rpm or
more).
[0057] Preferably, the formulations of the present invention are
stable under more than one (such as two, three, four, five, six or
seven) of the above stress conditions, most preferably under all of
the above stress conditions.
[0058] Accordingly, the polypeptide of the invention present in the
formulation of the invention: [0059] is stable after multiple (up
to 10) freeze/thaw cycles, said stability as determined by
OD320/OD280 measurement, SE-HPLC, RP-HPLC, IEX-HPLC, potency assay
(such as Biacore or ELISA) and/or SDS-PAGE; [0060] is stable during
storage at a temperature of 2-8.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more), said stability as
determined by OD320/OD280 measurement, SE-HPLC, RP-HPLC, IEX-HPLC,
potency assay (such as Biacore or ELISA) and/or SDS-PAGE; [0061] is
stable during storage at a temperature of 25.+-.5.degree. C. up to
at least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more), said
stability as determined by OD320/OD280 measurement, SE-HPLC,
RP-HPLC, IEX-HPLC, potency assay (such as Biacore or ELISA) and/or
SDS-PAGE; [0062] is stable during storage at a temperature of
37.+-.5.degree. C. up to at least 2 weeks (preferably at least 3
weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more), said stability as determined by OD320/OD280
measurement, SE-HPLC, RP-HPLC, IEX-HPLC, potency assay (such as
Biacore or ELISA) and/or SDS-PAGE; [0063] is stable when shaking
the formulation during 10 s to 1 min; [0064] is stable when pushing
the formulation through a needle (250, preferably 26 G, more
preferably 27 G, even more preferably 280, most preferably 290 or
more) with a syringe (the syringe used can be any commercially
available syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10
mL, 20 mL, 30 mL, 40 mL up to 50 mL syringe); [0065] is stable when
rotating for two days at 10 rpm; and/or [0066] is stable when
stirring for 1 hour at room temperature and/or 4-48 hours (such as
4-8 hours, 12 hours, 24 hours or even 48 hours) at 4.degree. C. at
at least 10 rpm (such as 50 rpm, 100 rpm or more).
[0067] The stability of the formulations of the present invention
can be demonstrated by the fact that less than 10% of the
polypeptides forms pyroglutamate at the N-terminal glutamic acid
(e.g. as assessed by RP-HPLC) and/or less than 10% of the
polypeptides forms dimers (e.g. as assessed by SE-HPLC) during
storage under one or more of the above stress conditions.
Preferably less than 10% of the polypeptides forms pyroglutamate at
the N-terminal glutamic acid (e.g. as assessed by RP-HPLC) and less
than 10% of the polypeptides forms dimers (e.g. as assessed by
SE-HPLC) during storage under one or more of the above stress
conditions.
[0068] In a specific aspect, less than 10% of the polypeptides
present in the formulation of the invention forms pyroglutamate at
the N-terminal glutamic acid (e.g. as assessed by RP-HPLC) during
storage at a temperature of 37.+-.5.degree. C. for up to at least 2
weeks (preferably at least 3 weeks, at least 5 weeks, at least 8
weeks, at least 10 weeks, at least 3 months, at least 6 months, at
least 1 year, 1.5 year or even 2 years or more). In another
specific aspect, less than 10% of the polypeptides forms dimers
(e.g. as assessed by SE-HPLC) during storage at a temperature of
37.+-.5.degree. C. for up to at least 2 weeks (preferably at least
3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more). In yet another specific aspect, less than
10% of the polypeptides present in the formulation of the invention
forms pyroglutamate at the N-terminal glutamic acid (e.g. as
assessed by RP-HPLC) during storage at a temperature of
37.+-.5.degree. C. for up to at least 2 weeks (preferably at least
3 weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more) and less than 10% of the polypeptides forms
dimers (e.g. as assessed by SE-HPLC) during storage at a
temperature of 37.+-.5.degree. C. for up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more).
[0069] Apart from this and/or in addition, the stability of the
formulations of the present invention can be demonstrated by the
fact that it shows only low to undetectable levels of aggregation
and/or particulate formation (e.g. as assessed by SE-HPLC,
subvisible particle counting, analytical ultracentrifugation,
dynamic light scattering, OD320/OD280 ratio measurement and/or
elastic light scattering) even during storage under one or more of
the above stress conditions. In a specific aspect, the formulations
of the present invention show only low to undetectable levels of
aggregation and/or particulate formation (e.g. as assessed by
SE-HPLC, subvisible particle counting, analytical
ultracentrifugation, dynamic light scattering and/or OD320/OD280
measurement) at a temperature of 37.+-.5.degree. C. and/or
5.+-.5.degree. C. for up to at least 2 weeks (preferably at least 3
weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more).
[0070] Apart from this and/or in addition, the stability of the
formulations of the present invention can be demonstrated by the
fact that it shows only low to undetectable levels of fragmentation
and/or degradation of the polypeptides (e.g. as assessed by
SDS-PAGE, SE-HPLC, RP-HPLC and/or IEX-HPLC) even during storage
under one or more of the above stress conditions. In a specific
aspect, the formulations of the present invention show only low to
undetectable levels of fragmentation and/or degradation of the
polypeptides (e.g. as assessed by SDS-PAGE, SE-HPLC, RP-HPLC and/or
IEX-HPLC) at a temperature of 37.+-.5.degree. C. for up to at least
2 weeks (preferably at least 3 weeks, at least 5 weeks, at least 8
weeks, at least 10 weeks, at least 3 months, at least 6 months, at
least 1 year, 1.5 year or even 2 years or more).
[0071] Apart from this and/or in addition, the stability of the
formulations of the present invention can be demonstrated by the
fact that it shows very little to no loss of the biological
activities of the polypeptide of the invention (e.g. as assessed by
ELISA and/or Biacore) even during storage under one or more of the
above stress conditions. In a specific aspect, the formulations of
the present invention show very little to no loss of the biological
activities of the polypeptide of the invention (e.g. as assessed by
ELISA and/or Biacore) at a temperature of 37.+-.5.degree. C. for up
to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,
at least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more).
[0072] More specifically, in the formulations of the present
invention at least 80% (preferably at least 90%, more preferably at
least 95% or even at least 99%) of the polypeptides retain their
binding activity to at least one (preferably to all) of their
targets (e.g. as assessed by ELISA and/or Biacore) after storage
under one or more of the above stress conditions compared to the
binding activity prior to storage.
[0073] In a specific aspect, at least 80% (preferably at least 90%,
more preferably at least 95% or even at least 99%) of the
polypeptides retains their binding activity (e.g. as assessed by
ELISA and/or Biacore) to at least one (preferably to all) of their
targets after storage at 37.+-.5.degree. C. for up to at least 2
weeks (preferably at least 3 weeks, at least 5 weeks, at least 2
months, at least 6 months, at least 1 year, 1.5 year or even 2
years or more) compared to the binding activity prior to
storage.
[0074] Accordingly the present invention provides stable
formulations of polypeptides comprising one or more single variable
domains, wherein: [0075] less than 10% of the polypeptides forms
pyroglutamate at the N-terminal glutamic acid (e.g. as assessed by
RP-HPLC) during storage at a temperature of 37.+-.5.degree. C. up
to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,
at least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more); [0076]
less than 10% of the polypeptides forms dimers (e.g. as assessed by
SE-HPLC) during storage at a temperature of 37.+-.5.degree. C. up
to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,
at least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more); [0077]
at least 80% of the polypeptides retain its binding activity (e.g.
as assessed by ELISA and/or Biacore) to at least one (preferably to
all) of its targets after storage at 37.+-.5.degree. C. up to 2
weeks (preferably at least 3 weeks, at least 5 weeks, at least 2
months, at least 6 months, at least 1 year, 1.5 year or even 2
years or more) compared to the binding activity prior to storage;
and/or [0078] the polypeptide is stable under mechanical
stress.
[0079] In a preferred aspect, the formulation of the invention is a
pharmaceutical formulation.
[0080] The present invention further provides methods for preparing
the stable formulations of the invention. The methods of the
invention may comprise the steps of concentrating a polypeptide
comprising one or more single variable domains and exchanging it
with the preferred buffer and/or excipient.
[0081] Also provided are containers, kits and pharmaceutical unit
dosages comprising the formulations of the invention for use by,
e.g., a healthcare professional. In specific embodiments, the kits
or pharmaceutical unit dosages comprising the stable formulations
of the invention are formulated for parenteral administration
(e.g., intradermally, intramuscularly, intraperitoneally,
intravenously and/or subcutaneously) of the polypeptide of the
invention to a human subject. The formulations, containers,
pharmaceutical unit dosages and/or kits can be used in prophylaxis
and/or therapy. In a specific aspect, the formulations, containers,
pharmaceutical unit dosages and/or kits are used for the prevention
and/or treatment of one or more diseases and/or disorders such as
bone diseases and/or disorders (such as e.g. osteoporosis,
cancer-related bone diseases, and/or bone loss associated with
autoimmunity and/or viral infection) or autoimmune diseases (such
as e.g. rheumatoid arthritis).
FIGURE LEGENDS
[0082] FIG. 1. The 280 nm SE-HPLC chromatograms of RANKL008a
formulated in phosphate (A), or histidine (8) buffers with either
50 mM NaCl, 100 mM NaCl or 10% mannitol, before and after 10
freeze/thaw cycles. A zoom on the main peak is shown as inset.
[0083] FIG. 2. The 280 nm RP-HPLC chromatograms of RANKL008a
formulated in phosphate buffers with either 50 mM NaCl, 100 mM NaCl
or 10% mannitol, before and after 10 freeze/thaw cycles. A zoom on
the main peak is shown as inset.
[0084] FIG. 3. The 280 nm SE-HPLC chromatograms of RANKL008a
formulated in phosphate buffer with either 50 mM NaCl, 100 mM NaCl
or 10% mannitol, after incubation for 2 weeks at 37.degree. C. A
zoom on the main peak is shown as inset.
[0085] FIG. 4. Figure demonstrating the time-dependent decrease (A)
and increase (B) of the surface area of, respectively, the main
peak (A) and % dimers (B) observed in SE-HPLC analysis of RANKL008a
formulated in different buffers and stored up to 10 weeks at
37.degree. C.
[0086] FIG. 5. The 280 nm RP-HPLC chromatograms of RANKL008a
formulated in phosphate buffer with either 50 mM NaCl, 100 mM NaCl
or 10% mannitol, after incubation for 2 weeks at 37.degree. C. A
zoom on the main peak is shown as inset.
[0087] FIG. 6. Overlay of the 280 nm IEX-HPLC chromatograms of
RANKL008a formulated in phosphate buffer with either 50 mM NaCl,
100 mM NaCl or 10% mannitol, after incubation for 2 weeks at
37.degree. C. A zoom on the main peak and postpeaks is shown as
inset.
[0088] FIG. 7. Relative amounts (%) of the main peak and the two
postpeaks observed in the IEX-HPLC chromatograms of the stability
samples after storage for 10 weeks at 37.degree. C.
[0089] FIG. 8. Picture and visual observation of the vials after
shaking. The RANKL008a sample, diluted (to 5 mg/mL) and undiluted
with or without 0.01% Tween 80 was shaken strongly (10 s-1
min).
[0090] FIG. 9. Overview of dilutions and steps made in
syringeability study as described in Example 1.6.
[0091] FIG. 10. OD 320/278 and OD 350/278 ratios (n=3) after
passage/storage of RANKL008a in syringes with different diluents as
described in Example 1.6.
[0092] FIG. 11. Relative HSA and RANKL potency of RANKL008a after
dilution in different diluents and passage/storage in syringes as
described in Example 1.6.
[0093] FIG. 12. Overview of needle/gauge size study with diluted
RANKL008a as described in Example 1.7.
[0094] FIG. 13. Overview of further needle/gauge size study with
diluted and undiluted RANKL008a as described in Example 1.7.
[0095] FIG. 14. Overview of the results obtained for thermal
stability testing of IL6R304 (A, C, E) and IL6R305 (B, D, F) and in
function of NaCl concentration (A, B), mannitol added (C, D) and
buffer/excipient (E, F).
[0096] FIG. 15. Overview of the results obtained in thermal
stability testing of IL6R304 in Tris buffer pH 7.2 or Histidine pH
6.5, with sucrose, glycine or mannitol added as excipient.
[0097] FIG. 16. Thermograms (obtained after subtracting the
base-lines) of IL6R304 in the buffers as indicated in the
graph.
[0098] FIG. 17. PAMAS analysis of IL6R304 formulated in PBS
compared to IL6R304 formulated in PBS+0.01% or 0.02% Tween 80.
Counts of particles: >10 .mu.m, >25 .mu.m, >50 .mu.m,
>100 .mu.m and >200 .mu.m.
[0099] FIG. 18. SE-HPLC for the IL6R304 molecule in the presence of
different concentrations of Tween 80,
[0100] FIG. 19. (A)-(C): Log-values of the soluble IL-6R Nanobody
concentration (Y-axis) vs. PEG6000 concentration (%) (X-axis),
together with the calculated solubility values. (D) represents an
example of how the linear regression analysis is performed on the
obtained data points to determine the intercept with the X-axis
(from which the theoretical solubility at zero % PEG6000 can be
deduced).
[0101] FIG. 20. Overlay of the SE-HPLC chromatograms of IL6R304
formulated at 10 mg/mL stored for 3 weeks at 37.degree. C. Inset,
zoom on the main peak to demonstrate the buffer-dependent
differences in % aggregates.
[0102] FIG. 21. Figure demonstrating the buffer-dependent
differences in % aggregates (peak surface area in SE-HPLC) that
were observed in the stability samples of IL6R304 and IL6R305
stored for 1 week at 37.degree. C.
[0103] FIG. 22, Figure demonstrating the buffer-dependent
differences in % aggregates (peak surface area in SE-HPLC) that
were observed in the stability samples of IL6R304 and IL6R305
stored for 3 weeks at 37.degree. C.
[0104] FIG. 23. Overlays of the RP-HPLC chromatograms from IL6R304
incubated for 3 weeks at 37.degree. C. in different formulation
buffers. A zoom on the main peak is shown as inset. Note the
increase of two minor prepeaks and a postpeak in the IL6R304
samples stored for 3 weeks.
[0105] FIG. 24. Osmolality data of IL6R304 (10 mg/mL) in the 12
different buffer indicated in the graph. The horizontal bar defines
the isotonic region.
[0106] FIG. 25. Figure demonstrating the time-dependent increase of
the % oligomers/aggregates (Y-axis) observed in SE-HPLC analysis of
IL6R304 stored for up to 5 weeks at 37.degree. C. (A) and 5.degree.
C. (B) in the buffers indicated in the graph. The %
oligomers/aggregates is expressed as the sum of the % peak surface
areas of prepeak1a, prepeak 1b and prepeak 2 relative to the total
peak surface area.
[0107] FIG. 26. (A): the % oligomers (% peak surface area) observed
in the Histidine buffers after storage for 3 weeks at 37.degree. C.
compared to the equivalent sample in PBS buffer. (B):
time-dependent and buffer-dependent increase in the % oligomers
observed in the IL-6R stability samples stored for up to 5 weeks at
37.degree. C., at a concentration of 10 mg/mL in the buffers
indicated in the graph.
[0108] FIG. 27. SE-HPLC profile of IL6R304 in the different buffers
as depicted in Table 21. IL6R304 was less prone to oligomerization
in L-histidine buffer compared to phosphate buffer. (A) Less
oligomers, which were seen as prepeaks during SE-HPLC analysis,
were present in IL6R304 samples stored for 8 weeks at 37.degree. C.
in L-histidine buffer (buffers 1-5) compared to phosphate buffer
(buffers 6-10). The amount of oligomers was lowest in buffer 3. (B)
The effect of the different excipients on oligomerization in either
L-histidine versus phosphate. Note that the surface area of the
postpeak was higher in phosphate compared to L-histidine.
[0109] FIG. 28. Kinetics of oligomer formation upon storage of
IL6R304 in the different buffers. Oligomerization was significantly
slower in L-histidine (buffers 1-5) compared to phosphate buffer
(buffers 6-10).
[0110] FIG. 29. Overlay of the RP-HPLC chromatograms from IL6R304
after storage for up to 8 weeks at +37.degree. C. in 10 different
formulation buffers. A zoom on the main peak and sidepeaks is shown
as inset.
[0111] FIG. 30. Kinetics of the formation for the pyroglutamate
variant of IL6R304 upon storage under stressed conditions in the
different buffers. Less pyroglutamate is formed in L-histidine
compared to phosphate buffer.
[0112] FIG. 31. Aggregation index before and after stirring of
IL6R304.
[0113] FIG. 32. Graphical presentation of Tm values for 23IL0064 as
a function of NaCl concentration (A) and of mannitol concentration
(B). Tm's were obtained in the thermal shift assay at 0.1
mg/mL.
[0114] FIG. 33. Graphical representation of the melting
temperatures measured for 2310075 as a function of the pH, in 10 mM
acetate, in 10 mM histidine, and in 10 mM phosphate buffer. The
protein concentration was 0.2 mg/mL. Scanning was performed at
1.degree. C./min, starting at 30.degree. C.
[0115] FIG. 34. SE-HPLC chromatograms (OD280) of 23IL0064 in 40 mM
histidine (pH 6.0)/50 mM NaCl. The start sample was compared to
samples after concentration. No aggregate formation due to
concentration of the sample in the histidine buffer was observed.
The SE-HPLC was run with a Phenomenex BioSep SEC S-2000 column,
with D-PBS as mobile phase at 0.2 mL/min.
[0116] FIG. 35. Log-values of the soluble protein concentration
(Y-axis) vs. PEG6000 concentration (%, X-axis). The right upper
panel represents an example of how the linear regression analysis
is performed on the obtained data points to determine the intercept
with the X-axis (from which the theoretical solubility at zero %
PEG6000 can be deduced). In the histidine buffer, the experiment
was performed at two concentrations which are presented in the two
bottom graphs. For these two graphs no regression was possible.
[0117] FIG. 36. Log-values of the soluble protein concentration
(Y-axis) of p23IL0064 and 23IL0075 in 10 mM phosphate buffer pH 7
with 50 mM NaCl vs. PEG6000 concentration (X-axis). By regression
analysis and extrapolation to a zero concentration of PEG6000, the
theoretical maximum protein concentrations (apparent solubility
values) were calculated and were for both proteins approximately 50
mg/mL. This number should only be used after confirmation with
other techniques.
[0118] FIG. 37. Log-values of the soluble protein concentration
(Y-axis) of p23IL0064 and p23IL0075 in 40 mM histidine pH 6.0
buffer with 50 mM NaCl vs. PEG6000 concentration (X-axis). In this
graph regression was not possible since no precipitation occurred
in the PEG % window explored.
[0119] FIG. 38. SE-HPLC chromatograms (280 nm) of 25 .mu.g 23IL0064
non-stressed and 37.degree. C. (3w and 4w)-stressed samples in
D-PBS. SE-HPLC was run on TSK-GEL G2000SWXL with D-PBS.
[0120] FIG. 39. RP-HPLC chromatograms (280 nm) of 25 .mu.g 23IL0064
non-stressed and 37.degree. C. (4w)-stressed sample in D-PBS.
RP-HPLC was run on Zorbax C-3 column with a water/acetonitrile 0.1%
TFA gradient.
[0121] FIG. 40. Graphical representation of the % peak area of the
different RP-HPLC peaks of 23IL0064 and product related substances
after 6 weeks stress at 37.degree. C. in different formulation
buffers. The first bar represents the reference sample, stored at
-80.degree. C. until analysis. The pre-peak 2 was present in the
start sample and did not increase during the storage at 37.degree.
C.
[0122] FIG. 41. Overlay of RP-HPLC chromatograms (280 nm) of
23IL0064 in 20 mM Histidine pH 6.5 (conc. 22.4 mg/mL). Comparison
of 6 weeks 37.degree. C., 6 weeks 25.degree. C., 6 weeks 4.degree.
C., and -80.degree. C. reference. RP-HPLC was run on Zorbax
300SB-C3 column with a water/acetonitrile 0.1% TFA gradient
0.3%/min) at 75.degree. C.
[0123] FIG. 42. SDS-PAGE of stability samples of 23IL0064 in 20 mM
Histidine pH 6.5 CONC stressed for 6 weeks at 4.degree. C.,
25.degree. C., 37.degree. C. and its -80.degree. C. Reference.
[0124] FIG. 43. Elastic light scattering as measured at 500 nm as a
function of the temperature for 23IL0075 samples with different
concentrations (as indicated in the legend).
[0125] FIG. 44. Elastic light scattering as measured at 500 nm as a
function of the temperature for 250 .mu.g/mL 23IL0075 in 10 mM
phosphate pH 6.0. The temperature of the onset of aggregate
formation is determined by means of linear fit.
[0126] FIG. 45. Elastic light scattering as measured at 500 nm as a
function of the temperature for 250 .mu.g/mL 23IL0075 in 10 mM
Acetate pH 6.0. The temperature of the onset of aggregate formation
is determined by means of linear fit.
[0127] FIG. 46. Elastic light scattering as measured at 500 nm as a
function of the temperature for 250 .mu.g/mL 23IL0075 in 10 mM
Histidine pH 6.0. The temperature of the onset of aggregate
formation is determined by means of linear fit.
[0128] FIG. 47. Graphical representation of the opalescence
(measured by OD500) (A) and the % oligomers (% pre-peak) detected
in SE-HPLC (B) after freeze/thaw stress. The percentage of
oligomers in the reference sample was approximately 0.4%. In this
study, an acetate, a histidine and a phosphate buffer were
compared, in combination with mannitol or a mixture of mannitol and
glycine as excipients, and Tween 80 or poloxamer in two different
concentrations as surfactants.
[0129] FIG. 48. Graphical representation of the opalescence
(measured by OD500) (A) and the % oligomers detected in SE-HPLC (B)
after shear stress. The percentage of oligomers in the reference
sample was approximately 0.4%. In this study, an acetate, a
histidine and a phosphate buffer were compared, in combination with
mannitol or a mixture of mannitol and glycine as excipients, and
Tween 80 or poloxamer in two different concentrations as
surfactants.
[0130] FIG. 49. Graphical representation of the opalescence
(measured by OD500) (A), and the % oligomers detected in SE-HPLC
(B) in different histidine buffers after freeze/thaw stress. The
percentage of oligomers in the reference sample was approximately
0.4%.*For one sample the condition without detergent was
included.
[0131] FIG. 50. Graphical representation of the opalescence
(measured by OD500) (A), the % oligomers detected in SE-HPLC (B),
and the % activity (albumin binding) of the Nanobody measured on
Biacore (C) after shear stress. The percentage of oligomers in the
reference sample was approximately 0.4%. In this study no
detergents were included, to mimic the situation during the final
concentration step of the DSP process.
[0132] FIG. 51. SE-HPLC chromatograms (280 nm) of 23IL0075 at 25
mg/mL in 10 mM Histidine pH 6.0, 10% sucrose, 0.005% Tween 80: the
reference sample compared to 6 weeks storage at 25.degree. C. and
37.degree. C.
DETAILED DESCRIPTION
[0133] Unless indicated or defined otherwise, all terms used have
their usual meaning in the art, which will be clear to the skilled
person. Reference is for example made to the standard handbooks,
such as Sambrook et al, "Molecular Cloning: A Laboratory Manual"
(2nd. Ed.), Vols. 1-3, Cold Spring Harbor Laboratory Press (1989);
F. Ausubel et al, eds., "Current protocols in molecular biology",
Green Publishing and Wiley Interscience, New York (1987); Lewin,
"Genes H", John Wiley & Sons, New York, N.Y., (1985); Old et
al., "Principles of Gene Manipulation: An Introduction to Genetic
Engineering", 2nd edition, University of California Press,
Berkeley, Calif. (1981); Roitt et al., "Immunology" (6th. Ed.),
Mosby/Elsevier, Edinburgh (2001); Roitt et al., Roitt's Essential
Immunology, 10th Ed. Blackwell Publishing, UK (2001); and Janeway
et al., "Immunobiology" (6th Ed.), Garland Science
Publishing/Churchill Livingstone, New York (2005), as well as to
the general background art cited herein.
[0134] As used herein, the term "isolated" in the context of a
polypeptide refers to a polypeptide which is substantially free of
cellular material or contaminating proteins from the cell or tissue
source from which it is derived, or substantially free of chemical
precursors or other chemicals when chemically synthesized. The
language "substantially free of cellular material" includes
preparations of a polypeptide in which the polypeptide is separated
from cellular components of the cells from which it is isolated or
recombinantly produced. Thus, a polypeptide that is substantially
free of cellular material includes preparations of a polypeptide
having less than about 30%, 20%, 10%, or 5% (by dry weight) of
heterologous protein, polypeptide, peptide, or antibody (also
referred to as a "contaminating protein"). When the polypeptide is
recombinantly produced, it may also be substantially free of
culture medium, i.e., culture medium represents less than about
20%, 10%, or 5% of the volume of the polypeptide preparation. When
the polypeptide is produced by chemical synthesis, it is preferably
substantially free of chemical precursors or other chemicals, i.e.,
it is separated from chemical precursors or other chemicals which
are involved in the synthesis of the polypeptide. Accordingly, such
preparations of a polypeptide have less than about 30%, 20%, 10%,
5% (by dry weight) of chemical precursors or compounds other than
the polypeptide of interest. In a specific embodiment, an
"isolated" polypeptide is purified by a multi-step purification
process that comprises two chromatography steps (e.g. cation
exchange and anion exchange), a 100K ultrafiltration step, followed
by a buffer exchange and concentration step in
Ultrafiltration/Diafiltration mode.
[0135] As used herein, the terms "subject" and "patient" are used
interchangeably. As used herein, the terms "subject" and "subjects"
refer to an animal, preferably a mammal including a non-primate
(e.g., a cow, pig, horse, cat, dog, rat, and mouse) and a primate
(e.g., a monkey, such as a cynomolgus monkey, chimpanzee, baboon
and a human), and more preferably a human. In a certain embodiment,
the subject is a mammal, preferably a human, with one or more
diseases or disorders, In another embodiment, the subject is a
mammal, preferably a human, at risk of developing one or more
diseases and/or disorders.
[0136] The terms "stability" and "stable" as used herein in the
context of a formulation comprising a polypeptide comprising one or
more single variable domains refer to the resistance of the
polypeptide in the formulation to aggregation, to the formation of
degradation products and/or to the formation of fragmentation
products under given transportation and/or storage conditions.
Apart from this and/or in addition, the "stable" formulations of
the invention retain biological activity under given transportation
and/or storage conditions. The stability of said polypeptide can be
assessed by degrees of aggregation, degradation and/or
fragmentation (as measured e.g. by SE-HPLC, RP-HPLC, IEX-HPLC,
subvisible particle counting, analytical ultracentrifugation,
dynamic light scattering, OD320/OD280 ratio measurement, elastic
light scattering, etc.), and/or by % of biological activity (as
measured e.g. by ELISA, Biacore, etc.) compared to a reference
formulation. For example, a reference formulation may be a
reference standard frozen at -20.degree. C. or below -65.degree. C.
(such as e.g. -80.degree. C.) consisting of the same polypeptide at
the same concentration in D-PBS or consisting of the same
polypeptide at the same concentration and in the same buffer as the
stressed samples but without applying the stress conditions, which
reference formulation regularly gives a single peak by SE-HPLC,
RP-HPLC and/or IEX-HPLC and/or keeps its biological activity in
Biacore and/or ELISA.
[0137] "Solubility" is often described as the maximum achievable
protein concentration whereby all of the protein remains in
solution. At this concentration the protein should still be
monomeric and free of aggregates. For determining protein
solubility only a limited number of (mostly empirical) techniques
are currently available. A first and popular technique consists of
concentrating the sample by using centrifugal ultrafiltration up to
the point where an opalescent solution is formed. Subsequently, the
insoluble fraction is removed and the protein content of the
supernatant is measured. Centrifugal concentrating devices such as
for example Vivaspin concentrators with a molecular weight cut-off
of 5 kDa can be used but require reasonable amounts of protein.
Solubility can also be monitored using an inert macromolecule such
as polyethylene glycol (PEG; Mr>6,000), which precipitates
proteins primarily through an excluded volume effect, a process
that can be generally understood in terms of a simple colloidal
phase separation. A logarithmic linear relationship between protein
solubility and weight percent polyethylene glycol can be obtained,
and from this plot the intercept yields the solubility value. The
term "good solubility" of the polypeptide of the invention, as used
herein, means that no or little precipitation is observed of the
polypeptide of the invention during downstream processing (DSP)
and/or during storage for a short or longer time at 5.degree. or
-20.degree. C. at concentrations ranging from 20-200 mg/ml or more.
The formation of precipitates (oligomers or other particulates) can
be measured e.g. by SE-HPLC, OD320/OD280 ratio measurement and/or
elastic light scattering. Preferably, the polypeptides present in
the formulations of the present invention have a solubility of at
least 20 mg/mL, at least 30 mg/mL, at least 40 mg/mL, at least 50
mg/mL, at least 60 mg/mL, at least 65 mg/mL, at least 70 mg/mL, at
least 80 mg/mL, at least 90 mg/mL, at least 100 mg/mL, at least 110
mg/mL, at least 120 mg/mL, at least 130 mg/mL, at least 140 mg/mL,
at least 150 mg/mL, at least 200 mg/mL or even more. Preferably,
the OD320/OD280 ratio of the formulations of the present invention
is 0.05 or lower, such as 0.01 or lower or 0.005 or lower. The
scattering in the formulation of the present invention should be
within detection limit and preferably lower than 1000 abs, such as
750 abs or lower or 500 abs or lower.
[0138] The phrase "low to undetectable levels of aggregation" as
used herein refers to samples containing no more than 5%, no more
than 4%, no more than 3%, no more than 2%, no more than 1% or no
more than 0.5% aggregation by weight of protein. Unless explicitly
referred to differently, aggregation as used in the present
invention means the development of high molecular weight
aggregates, i.e. aggregates with an apparent molecular weight of
more/higher than the apparent molecular weight observed in SE-HPLC
analysis for dimers of the polypeptide of the invention (such as
e.g. 44 kDa as observed for SEQ ID NO: 4; 36-38 kDa as observed for
SEQ ID NO's 1-3; and 36 kDa as observed for SEQ ID NO: 5 in
SE-HPLC) in comparison with molecular weight markers. Aggregation
can be assessed by various methods known in the art. Without being
limiting, examples include high performance size exclusion
chromatography (SE-HPLC), subvisible particle counting, analytical
ultracentrifugation (AUC), dynamic light scattering (DLS), static
light scattering (SLS), elastic light scattering, OD320/OD280
measurement, Fourier Transform infrared Spectroscopy (FTIR),
circular dichroism (CD), urea-induced protein unfolding techniques,
intrinsic tryptophan fluorescence and/or differential scanning
calorimetry techniques.
[0139] The term "low to undetectable levels of fragmentation and/or
degradation" as used herein refers to samples containing equal to
or more than 80%, 85%, 90%, 95%, 98% or 99% of the total protein,
for example, in a single peak as determined by SE-HPLC, RP-HPLC
and/or IEX-HPLC, representing the non-degraded polypeptide, and
containing no other single peaks having more than 5%, more than 4%,
more than 3%, more than 2%, more than 1%, or more than 0.5% of the
total protein in each.
[0140] The term "very little to no loss of the biological
activities" as used herein refers to single variable domain
activities, including but not limited to, specific binding
abilities of the single variable domain to the target of interest
as measured by various immunological assays, including, but not
limited to ELISAs and/or by Surface Plasmon Resonance (Biacore), In
one embodiment, the single variable domains of the formulations of
the invention retain at least 50%, preferably at least 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or even 99% or more of the
ability to specifically bind to an antigen as compared to a
reference formulation, as measured by an immunological assay known
to one of skill in the art or described herein. For example, an
ELISA based assay (e.g. as described in the Example section) may be
used to compare the ability of the single variable domain to
specifically bind to its target. A "reference formulation" as used
herein refers to a formulation that is frozen at a temperature of
-20.+-.5.degree. C. or at below -64.degree. C. (such as e.g. at
-80.degree. C.) consisting of the same single variable domain at
the same concentration in D-PBS or consisting of the same single
variable domains at the same concentration in the same
buffer/excipients as the stressed samples but without applying the
stress conditions, which reference formulation regularly gives a
single peak by SE-HPLC, RP-HPLC and/or IEX-HPLC and/or keeps its
biological activity in Biacore and/or ELISA.
[0141] The phrase "pharmaceutically acceptable" as used herein
means approved by a regulatory agency of the Federal or a state
government, or listed in the U.S. Pharmacopeia, European
Pharmacopoeia or other generally recognized pharmacopeia for use in
animals, and more particularly in humans. In this sense, it should
be compatible with the other ingredients of the formulation and not
eliciting an unacceptable deleterious effect in the subject.
[0142] According to the European Pharmacopoeia, a solution is
considered isotonic if it has an osmolality of 290.+-.30 mOsm/kg.
Osmolality measurements were therefore performed on the different
formulations used in the stability studies. Isotonicity can be
measured by, for example, a vapor pressure or ice-freezing type
osmometer.
[0143] As used herein, the term "effective amount" refers to the
amount of an agent (e.g. a prophylactic or therapeutic agent) which
is sufficient to reduce and/or ameliorate the severity and/or
duration of one or more diseases and/or disorders.
[0144] The term "polyol" as used herein refers to sugars that
contains many hydroxyl (--OH) groups compared to a normal
saccharide. Polyols include alcohols and carbohydrates such as
mannitol, sorbitol, maltitol, xylitol, isomalt, erythritol,
lactitol, sucrose, glucose, galactose, fructose, fucose, ribose,
lactose, maltose and cellubiose.
[0145] As used herein, the terms "therapeutic agent" and
"therapeutic agents" refer to any agent(s) which can be used in the
prevention, treatment and/or management of one or more diseases
and/or disorders. In the context of the present invention, the term
"therapeutic agent" refers to a polypeptide comprising one or more
single variable domains. In certain other embodiments, the term
"therapeutic agent" refers to an agent other than the polypeptide
of the invention which might be used in the formulation.
[0146] As used herein, the term "therapeutically effective amount"
refers to the amount of a therapeutic agent (e.g. a polypeptide
comprising one or more single variable domains), that is sufficient
to reduce the severity of one or more diseases and/or
disorders.
[0147] The term "excipient" as used herein refers to an inert
substance which is commonly used as a diluent, vehicle,
preservative, binder or stabilizing agent for drugs which imparts a
beneficial physical property to a formulation, such as increased
protein stability, increased protein solubility, and/or decreased
viscosity. Examples of excipients include, but are not limited to,
proteins (e.g., serum albumin), amino acids (e.g., aspartic acid,
glutamic acid, lysine, arginine, glycine), surfactants (e.g., SDS,
Tween 20, Tween 80, poloxamers, polysorbate and nonionic
surfactants), saccharides (e.g., glucose, sucrose, maltose and
trehalose), polyols (e.g., mannitol and sorbitol), fatty acids and
phospholipids (e.g., alkyl sulfonates and caprylate). For
additional information regarding excipients, see Remington's
Pharmaceutical Sciences (by Joseph P. Remington, 18th ed., Mack
Publishing Co., Easton, Pa.), which is incorporated herein in its
entirety.
[0148] The term "variable domain" or "immunoglobulin variable
domain" refers to the part or domain of an immunoglobulin molecule
or antibody which is partially or fully responsible for antigen
binding. The term "single variable domain" or "immunoglobulin
single variable domain" (both terms are used interchangeably),
defines molecules wherein the antigen binding site is present on,
and formed by, a single immunoglobulin domain. This sets single
variable domains apart from "conventional" immunoglobulins or their
fragments, wherein two immunoglobulin domains, in particular two
"variable domains" interact to form an antigen binding site.
Typically, in conventional immunoglobulins, a heavy chain variable
domain (VH) and a light chain variable domain (VL) interact to form
an antigen binding site. In this case, the complementarity
determining regions (CDRs) of both VH and VL will contribute to the
antigen binding site, i.e. a total of 6 CDRs will be involved in
antigen binding site formation.
[0149] In contrast, the binding site of a single variable domain is
formed by a single VH or VL domain. Hence, the antigen binding site
of a single variable domain is formed by no more than three CDRs.
The term "single variable domain" does comprise fragments of
conventional immunoglobulins wherein the antigen binding site is
formed by a single variable domain.
[0150] Generally, single variable domains will be amino acid
sequences that essentially consist of 4 framework regions (FR1 to
FR4 respectively) and 3 complementarity determining regions (COR1
to CDR3 respectively); or any suitable fragment of such an amino
acid sequence (which will then usually contain at least some of the
amino acid residues that form at least one of the CDR's). Such
single variable domains and fragments are most preferably such that
they comprise an immunoglobulin fold or are capable for forming,
under suitable conditions, an immunoglobulin fold. As such, the
single variable domain may for example comprise a light chain
variable domain sequence (e.g. a V, sequence) or a suitable
fragment thereof; or a heavy chain variable domain sequence (e.g. a
V.sub.L sequence or V.sub.HH sequence) or a suitable fragment
thereof; as long as it is capable of forming a single antigen
binding unit (i.e. a functional antigen binding unit that
essentially consists of the single variable domain, such that the
single antigen binding domain does not need to interact with
another variable domain to form a functional antigen binding unit,
as is for example the case for the variable domains that are
present in for example conventional antibodies and scFv fragments
that need to interact with another variable domain--e.g. through a
V.sub.H/V.sub.L interaction--to form a functional antigen binding
domain).
[0151] In one aspect of the invention, the single variable domains
are light chain variable domain sequences (e.g. a V.sub.L
sequence), or heavy chain variable domain sequences (e.g. a V.sub.H
sequence); more specifically, the single variable domains can be
heavy chain variable domain sequences that are derived from a
conventional four-chain antibody or heavy chain variable domain
sequences that are derived from a heavy chain antibody.
[0152] The single variable domain may be a domain antibody (or an
amino acid sequence that is suitable for use as a domain antibody),
a single domain antibody (or an amino acid sequence that is
suitable for use as a single domain antibody), a "dAb" (or an amino
acid sequence that is suitable for use as a dAb) or a Nanobody.RTM.
(as defined herein, and including but not limited to a V.sub.HH
sequence) [Note: Nanobody.RTM. and Nanobodies.RTM. are registered
trademarks of Ablynx N.V.]; other single variable domains, or any
suitable fragment of any one thereof. For a general description of
(single) domain antibodies, reference is also made to the prior art
cited herein, as well as to EP 0 368 684. For the term "dAb's",
reference is for example made to Ward et al. 1989 (Nature 341
(6242): 544-546), to Holt et al. 2003 (Trends Biotechnol. 21(11):
484-490); as well as to for example WO 04/068820, WO 06/030220, WO
06/003388 and other published patent applications of Domantis Ltd.
It should also be noted that, although less preferred in the
context of the present invention because they are not of mammalian
origin, single variable domains can be derived from certain species
of shark (for example, the so-called "IgNAR domains", see for
example WO 05/18629).
[0153] In particular, the polypeptides of the invention may
comprise one or more Nanobodies or a suitable fragment thereof. For
a further description of V.sub.HH's and Nanobodies, reference is
made to the review article by Muyldermans 2001 (Reviews in
Molecular Biotechnology 74: 277-302); as well as to the following
patent applications, which are mentioned as general background art:
WO 94/04678, WO 95/04079 and WO 96/34103 of the Vrije Universiteit
Brussel; WO 94/25591, WO 99/37681, WO 00/40968, WO 00/43507, WO
00/65057, WO 01/40310, WO 01/44301, EP 1 134 231 and WO 02/48193 of
Unilever; WO 97/49805, WO 01/21817, WO 03/035694, WO 03/054016 and
WO 03/055527 of the Vlaams instituut voor Biotechnologie (VIB); WO
03/050531 of Algonomics N.V. and Ablynx N.V.; WO 01/90190 by the
National Research Council of Canada; WO 03/025020 EP 1 433 793) by
the institute of Antibodies; as well as WO 04/041867, WO 04/041862,
WO 04/041865, WO 04/041863, WO 04/062551, WO 05/044858, WO
06/40153, WO 06/079372, WO 06/122786, WO 06/122787 and WO
06/122825, by Ablynx N.V. and the further published patent
applications by Ablynx N.V. Reference is also made to the further
prior art mentioned in these applications, and in particular to the
list of references mentioned on pages 41-43 of the International
application WO 06/040153, which list and references are
incorporated herein by reference. As described in these references,
Nanobodies (in particular V.sub.HH sequences and partially
humanized Nanobodies) can in particular be characterized by the
presence of one or more "Hallmark residue" in one or more of the
framework sequences. A further description of the Nanobodies,
including humanization and/or camelization of Nanobodies, as well
as other modifications, parts or fragments, derivatives or
"Nanobody fusions", multivalent constructs (including some
non-limiting examples of linker sequences) and different
modifications to increase the half-life of the Nanobodies and their
preparations can be found e.g. in WO 08/101,985 and WO
08/142,164.
[0154] The total number of amino acid residues in a Nanobody can be
in the region of 110-120, is preferably 112-115, and is most
preferably 113. It should however be noted that parts, fragments,
analogs or derivatives (as further described herein) of a Nanobody
are not particularly limited as to their length and/or size, as
long as such parts, fragments, analogs or derivatives meet the
further requirements outlined herein and are also preferably
suitable for the purposes described herein.
[0155] Thus, in the meaning of the present invention, the term
"single variable domain" comprises polypeptides which are derived
from a non-human source, preferably a camelid, preferably a camelid
heavy chain antibody. They may be humanized, as previously
described. Moreover, the term comprises polypeptides derived from
non-camelid sources, e.g. mouse or human, which have been
"camelized", as previously described.
[0156] The term "single variable domain" also encompasses variable
domains of different origin, comprising mouse, rat, rabbit, donkey,
human and camelid variable domains; as well as fully human,
humanized or chimeric variable domains. For example, the invention
comprises camelid variable domains and humanized camelid variable
domains, or camelized variable domains, e.g. camelized dAb as
described by Ward et al (see for example WO 94/04678 and Davies and
Riechmann (1994, FEBS Lett. 339(3): 285-290) and (1996, Protein
Eng. 9(6): 531-537)). Moreover, the invention comprises fused
variable domains, e.g. multivalent and/or multispecific constructs
(for multivalent and multispecific polypeptides containing one or
more V.sub.HH domains and their preparation, reference is also made
to Conrath et al. 2001 (J. Biol. Chem. 276: 7346-7350) as well as
to for example WO 96/34103 and WO 99/23221).
[0157] Unless indicated otherwise, the term "immunoglobulin
sequence"--whether used herein to refer to a heavy chain antibody
or to a conventional 4-chain antibody--is used as a general term to
include both the full-size antibody, the individual chains thereof,
as well as all parts, domains or fragments thereof (including but
not limited to antigen-binding domains or fragments such as
V.sub.HH domains or V.sub.H/V.sub.L domains, respectively). The
terms antigen-binding molecules or antigen-binding protein are used
interchangeably with immunoglobulin sequence, and include
Nanobodies.
[0158] The single variable domains provided by the invention are
preferably in essentially isolated form (as defined herein), or
form part of a polypeptide of the invention (as defined herein),
which may comprise or essentially consist of one or more single
variable domains and which may optionally further comprise one or
more further amino acid sequences (all optionally linked via one or
more suitable linkers). For example, and without limitation, the
one or more single variable domains may be used as a binding unit
in such a polypeptide, which may optionally contain one or more
further amino acid sequences that can serve as a binding unit (i.e.
against one or more other targets), so as to provide a monovalent,
multivalent or multispecific polypeptide of the invention,
respectively as e.g. described in WO 08/101,985, WO 08/142,164, WO
09/068,625, WO 09/068,627 and WO 08/020,079. Such a protein or
polypeptide may also be in essentially isolated form (as defined
herein) and the methods of the present invention for the expression
and/or production of single variable domains equally apply to
polypeptides comprising one or more single variable domains.
[0159] According to the invention, the term "single variable
domain" may comprise constructs comprising two or more antigen
binding units in the form of single variable domain, as outlined
above. For example, two (or more) variable domains with the same or
different antigen specificity can be linked to form e.g. a
bivalent, trivalent or multivalent construct. By combining variable
domains of two or more specificities, bispecific, trispecific etc.
constructs can be formed. For example, a variable domain according
to the invention may comprise two variable domains directed against
target A, and one variable domain against target B. Such constructs
and modifications thereof, which the skilled person can readily
envisage, are all encompassed by the term variable domain as used
herein and are also referred to as "polypeptide of the invention"
or "polypeptides of the invention".
[0160] As further described in paragraph m) on page 53 of WO
08/020,079, an amino acid sequence (such as a Nanobody, an
antibody, a polypeptide of the invention, or generally an antigen
binding protein or polypeptide or a fragment thereof) that can
(specifically) bind to, that has affinity for and/or that has
specificity for a specific antigenic determinant, epitope, antigen
or protein (or for at least one part, fragment or epitope thereof)
is said to be "against" or "directed against" said antigenic
determinant, epitope, antigen or protein.
[0161] The polypeptide comprising one or more single variable
domains for use in the formulation of the invention may be
therapeutic or prophylactic, and may be useful in the treatment
and/or management of one or more diseases. In one specific aspect,
the polypeptide has at least two single variable domains. In
another specific aspect, the polypeptide has at least three single
variable domains. Preferably, the polypeptide comprises at least
one single variable domain directed against HSA. In another
specific aspect, the polypeptide comprises at least a single
variable domain against RANKL. In another specific aspect, the
polypeptide comprises at least a single variable domain against
IL-6R. In another specific aspect, the polypeptide comprises at
least a single variable domain against IL-23. More preferably, the
polypeptide is directed against and/or specifically binds RANKL and
HSA, IL-6R and HSA and/or IL-23 and HSA. In yet another aspect,
polypeptide comprises at least a single variable domain against
RANKL and at least a single variable domain against HSA. In yet
another aspect, polypeptide comprises at least a single variable
domain against IL-6R and at least a single variable domain against
HSA. In yet another aspect, polypeptide comprises at least a single
variable domain against IL-23 and at least a single variable domain
against HSA. In yet another aspect, polypeptide comprises at least
two single variable domains against RANKL and at least a single
variable domain against HSA. In yet another aspect, polypeptide
comprises at least two single variable domains against IL-6R and at
least a single variable domain against HSA. In yet another aspect,
polypeptide comprises at least two single variable domains against
IL-23 and at least a single variable domain against HSA. In a
preferred aspect, the single variable domains used in the
polypeptide of the invention are selected from WO 08/142,164 (such
as e.g. SEQ ID NO's: 745 and/or 791 of WO 08/142,164), WO
08/020,079, WO 09/068,627 (such as e.g. SEQ ID NO's 2578, 2584
and/or 2585 of WO 09/068,627), PCT application No.
PCT/EP2010/054747 by Ablynx N.V., POT application No.
PCT/EP2010/054764 by Ablynx N.V. (such as e.g. SEQ ID NO's: 66
and/or 98 of PCT/EP2010/054764) and WO 08/028,977 (such as e.g. SEQ
ID NO: 62 of WO 08/028,977). Preferred polypeptides of the
invention are selected from SEQ ID NO's: 1 to 6.
[0162] The concentration of polypeptide of the invention present in
the formulation can by any concentration of the polypeptide that
provides the desired effect to the subject. In a preferred aspect,
the concentration of the polypeptide of the invention is from 1 to
200 mg/mL such as about 1 mg/mL, about 2 mg/mL, about 5 mg/mL,
about 10 mg/mL, about 15 mg/mL or about 20 mg/mL, about 30 mg/mL,
about 40 mg/mL, about 50 mg/mL, about 60 mg/mL, about 65 mg/mL,
about 70 mg/mL, about 80 mg/mL, about 90 mg/mL or about 100 mg/mL
or more. In certain embodiments, the concentration of polypeptide
of the invention can be 110 mg/mL or more, 120 mg/mL or more, 130
mg/mL or more, 140 mg/mL or more, 150 mg/mL or more or even 200
mg/mL or more. In a specific aspect, a formulation of the invention
comprises about 10 mg/mL of polypeptide of the invention.
[0163] The formulation of the invention comprises an aqueous
carrier having a pH of 5.5 to 8.0 and a polypeptide as defined
above ("polypeptide of the invention") comprising one or more
single variable domains at a concentration of 1 mg/mL to 200 mg/mL,
said formulation being formulated for administration to a human
subject, wherein said formulation further comprises one or more
components selected from: [0164] a) A buffer at a concentration of
10 mM to 100 mM selected from the group consisting of histidine pH
6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and
acetate pH 5.5-6.0; [0165] b) An excipient at a concentration of 1%
to 20%; [0166] c) A surfactant at a concentration of 0.001% to 1%
selected from Tween 80, Tween 20 or a poloxamer; and wherein said
formulation has an inorganic salt concentration of 150 mM or
lower.
[0167] The stable formulations of the present invention comprise
polypeptides of the invention that have a good solubility and a
high stability even during transportation and/or long periods of
storage and that exhibit little to no aggregation. In addition to
the polypeptide of the invention, the formulations of the present
invention comprise at least an aqueous carrier and a buffer. The
carrier used in the formulation of the invention should be a liquid
carrier. Preferably the carrier is an aqueous carrier such as e.g.
distilled water, MilliQ water or Water for Injection (WFI).
[0168] The formulation should not contain inorganic salt at a
concentration of more than 150 mM. Without being limiting,
inorganic salts for use in the formulation of the invention can be
selected from NaCl and KC. Accordingly the formulation of the
invention has an inorganic salt concentration of 150 mM or lower,
preferably 120 mM or lower, or 100 mM or lower, more preferably 90
mM or lower, 80 mM or lower, 75 mM or lower, such as 50 mM or lower
or even 40 mM or lower, 25 mM or lower, 10 mM or lower or 5 mM or
lower. Most preferably, the formulation does not contain any
inorganic salt.
[0169] The pH of the formulation of the invention generally should
not be equal to the isoelectric point of the particular polypeptide
of the invention present in the formulation and may range from
about 5.5 to about 8.0, or from about 6.0 to about 7.5, preferably
from about 6.2 to 7.5, from about 6.5 to 7.5, most preferably from
about 6.5 to 7.0. In a specific aspect, the formulation of the
invention has a pH of about 6.5. In another specific aspect, the
formulation of the invention has a pH of about 7.0. In yet another
specific aspect, the formulation of the invention has a pH of about
6.0.
[0170] The formulation may be buffered by a buffer selected from
the group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES
pH 6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0, preferably
hepes pH 7.0 or histidine pH 6.0-6.5, such as histidine pH 6.5 or
histidine pH 6.0.
[0171] The concentration of the buffer present in the formulation
of the invention may range from 1 mM to 100 mM, 5 mM to 100 mM, 5
mM to 75 mM, 5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 25 mM, 10 mM
to 20 mM. In a specific aspect, the concentration of buffer in the
formulations of the invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20
mM, 25 mM, 50 mM, 75 mM, or 100 mM. Preferably, the concentration
is between 10 and 20 mM, such as 10 mM or 15 mM.
[0172] Any form of histidine suitable for formulation and
parenteral administration may be used in the formulation of the
invention. The purity of histidine should be at least 98%, at least
99%, or at least 99.5%. In a specific aspect, a formulation of the
invention comprises 15 mM histidine buffer pH 6.5. In another
specific aspect, a formulation of the invention comprises 10 mM
histidine buffer pH 6.0.
[0173] Apart from or in addition to histidine, hepes buffers pH7.0
may be used in the formulations of the present invention. Any form
of hepes suitable for formulation and parenteral administration may
be used in the formulation of the invention. The purity of hepes
should be at least 98%, at least 99%, or at least 99.5%. In a
specific aspect, a formulation of the invention comprises 15 mM
hepes buffer pH7.0.
[0174] It will be understood by one skilled in the art that the
formulation of the invention may be isotonic or slightly hypotonic
with human blood, i.e. the formulation of the invention has
essentially the same or a slightly lower osmotic pressure as human
blood. Such isotonic or slightly hypotonic formulation generally
has an osmotic pressure from about 240 mOSm/kg to about 320
mOSm/kg, such as about 240 mOSm/kg or higher, 250 mOSm/kg or higher
or 260 mOSm/kg or higher.
[0175] Tonicity of a formulation is adjusted by the use of tonicity
modifiers. "Tonicity modifiers" are those pharmaceutically
acceptable inert substances that can be added to the formulation to
provide an isotonicity of the formulation. A preferred tonicity
modifier in the formulation of the invention are excipients.
Preferred excipients for use in the formulation of the invention
may be selected from sugars, polyols and surfactants.
[0176] Accordingly, in another aspect, the formulation of the
invention comprises an excipient. Preferred excipients include
polyols and/or sugars. The polyol and/or sugar may be a
monosaccharide such as glucose or mannose, or a polysaccharide
including disaccharides such as (without being limiting) sucrose
and lactose, as well as sugar derivatives including sugar alcohols
and sugar acids. Polyols and sugar alcohols include (without being
limiting) mannitol, xylitol, erythritol, threitol, sorbitol and
glycerol. A non-limiting example of a sugar acid is L-gluconate.
Other exemplary sugars include (without being limiting) trehalose,
glycine, maltose, raffinose, etc. The concentration of the
excipient may range from about 1% to 10% (w:v), preferably from
about 2.5% to 10% (w:v), more preferably from about 5% to 10%
(w:v), such as e.g. 5% (w:v), 7.5% (w:v), 8% or 10% (w:v).
Throughout the present invention the concentration of the excipient
will be given as % (w:v). In a preferred aspect, the formulation
comprises sucrose, preferably at a concentration of about 5% to 10%
(w:v), such as about 8% (w:v).
[0177] In another aspect, the formulation of the invention
comprises a surfactant. A surfactant refers to a surface-active
agent comprising a hydrophobic portion and a hydrophilic portion.
In a preferred aspect, the surfactant is non-ionic. Certain
exemplary non-ionic surfactants include (without being limiting)
PEG8000, and polysorbate, including without being limiting,
polysorbate 80 (Tween 80) and polysorbate 20 (Tween 20), Triton
X-100, polyoxypropylene-polyoxyethylene esters (Pluronic.RTM.), and
NP-40. In a specific aspect, the surfactant is selected from Tween
20, Tween 80 or a poloxamer. The concentration of the surfactant
may range from about 0.001% to 1% (v:v) (preferably from about
0.001% to 0.1% (v:v), or 0.01% to 0.1% (v:v) such as 0.001% (v:v),
0.005% (v:v), 0.01% (v:v), 0.02% (v:v), 0.05% (v:v), 0.08% (v:v),
0.1% (v:v), 0.5% (v:v), or 1% (v:v) of the formulation, preferably
0.01% (v:v)). Throughout the present invention the concentration of
the surfactant will be given as % (v:v). In a specific embodiment,
the surfactant is Tween 20 or Tween 80, which is at a concentration
of 0.001% (v:v), 0.005% (v:v), 0.01% (v:v), 0.02% (v:v), 0.05%
(v:v), 0.08% (v:v), 0.1% (v:v), 0.5% (v:v) or 1% (v:v) of the
formulation, preferably 0.01% (v:v).
[0178] In a preferred aspect, the formulation of the present
invention comprises an aqueous carrier having a pH of 5.5 to 8.0
and a polypeptide as defined above ("polypeptide of the invention")
comprising one or more single variable domains at a concentration
of 1 mg/mL to 200 mg/mL, said formulation being formulated for
administration to a human subject and said formulation further
comprises at least two components selected from: [0179] a) A buffer
at a concentration of 10mM to 100 mM selected from the group
consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,
succinate pH 6.0-6.5 and acetate pH 5.5-6.0; [0180] b) An excipient
at a concentration of 1% to 20%; [0181] c) A surfactant at a
concentration of 0.001% to 1% selected from Tween 80, Tween 20 or
poloxamers; wherein said formulation has an inorganic salt
concentration of 150 mM or lower.
[0182] In one aspect, in addition to the polypeptide of the
invention, the formulation of the present invention may comprise at
least an aqueous carrier having a pH of 5.5 to 8.0, a buffer
selected from the group consisting of histidine pH 6.0-6.5, hepes
pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0, and an excipient. Preferably the buffer is histidine pH
6.0-6.5 or hepes pH 7.0. Most preferably the buffer is histidine pH
6.5 or histidine pH 6.0. The buffer preferably has a concentration
ranging from 1 mM to 100 mM, 5 mM to 100 mM, 5 mM to 75 mM, 5 mM to
50 mM, 10 mM to 50 mM, 10 mM to 25 mM, 10 mM to 20 mM. In a
specific aspect, the concentration of buffer in the formulations of
the invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50
mM, 75 mM, or 100 mM. In a preferred aspect, the concentration is
between 10 and 20 mM, such as 10 mM or 15 mM. In a specific aspect,
a formulation of the invention comprises 15 mM histidine buffer pH
6.5. In another specific aspect, a formulation of the invention
comprises 10 mM histidine buffer pH 6.0. Preferred excipients are
polyols such as mannitol, sorbitol, etc., saccharides such as e.g.
sucrose, mannose, trehalose, etc. The concentration of the
excipient may range from about 1% to 20%, preferably from about
2.5% to 15%, more preferably from about 5% to 10%, such as e.g. 5%,
7.5%, 8% or 10%. In a preferred aspect, the formulation comprises
sucrose, preferably at a concentration of about 5% to 10%, such as
about 8% or about 10%. Accordingly, a preferred formulation of the
invention may comprise 15 mM histidine pH 6.5 and 8% sucrose.
Another preferred formulation of the invention may comprise 10 mM
histidine pH 6.0 and 10% sucrose.
[0183] In another aspect, in addition to the polypeptide of the
invention, the formulation of the present invention may comprise at
least an aqueous carrier having a pH of 5.5 to 8.0, a buffer
selected from the group consisting of histidine pH 6.0-6.5, hepes
pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0, and a surfactant. Preferably the buffer is selected from
histidine pH 6.0-6.5 or hepes pH 7.0. Most preferably the buffer is
histidine pH 6.5 or histidine pH 6.0. The buffer preferably has a
concentration ranging from 1 mM to 100 mM, 5 mM to 100 mM, 5 mM to
75 mM, 5 mM to 50 mM, 10 mM to 50 mM, 10 mM to 25 mM, 10 mM to 20
mM. In a specific aspect, the concentration of buffer in the
formulation of the invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20
mM, 25 mM, 50 mM, 75 mM, or 100 mM. In a preferred aspect, the
concentration is between 10 and 20 mM, such as 10 mM or 15 mM. In a
specific aspect, a formulation of the invention comprises 15 mM
histidine buffer pH 6.5. In another specific aspect, a formulation
of the invention comprises 10 mM histidine buffer pH 6.0. The
surfactant may be selected from Tween 20, Tween or poloxamers. The
concentration of the surfactant may range from about 0.001% to 1%
(preferably from about 0.001% to 0.1%, or 0.01% to 0.1% such as
0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of
the formulation, preferably 0.01%). In a specific embodiment, the
surfactant is Tween 20 or Tween 80, which is at a concentration of
0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5% or 1% of the
formulation, such as e.g. 0.01% Tween 80 or 0.005% Tween 80.
Accordingly, a preferred formulation of the invention may comprise
15 mM histidine pH 6.5 and 0.01% Tween 80. Another preferred
formulation of the invention may comprise 10 mM histidine pH 6.0
and 0.005% Tween 80.
[0184] In yet another aspect, in addition to the polypeptide of the
invention, the formulation of the present invention may comprise at
least an aqueous carrier having a pH of 5.5 to 8.0, an excipient
and a surfactant. Preferred excipients are polyols such as
mannitol, sorbitol, etc., saccharides such as e.g. sucrose,
mannose, trehalose, etc. The concentration of the excipient may
range from about 1% to 20%, preferably from about 2.5% to 15%, more
preferably from about 5% to 10%, such as e.g. 5%, 7.5%, 8% or 10%.
In a preferred aspect, the formulation comprises sucrose,
preferably at a concentration of about 5% to 10%, such as about 8%
or 10%. The surfactant may be selected from Tween 20, Tween 80 or a
poloxamer. The concentration of the surfactant may range from about
0.001% to 1% (preferably from about 0.001% to 0.1%, or 0.01% to t
0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%,
0.5%, or 1% of the formulation, preferably 0.01%). In a specific
embodiment, the surfactant is Tween 20 or Tween 80, which is at a
concentration of 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%,
0.5% or 1% of the formulation, such as e.g. 0.01% Tween 80 or
0.005% Tween 80. Accordingly, a preferred formulation of the
invention may comprise 8% sucrose and 0.01% Tween 80. Another
preferred formulation of the invention may comprise 10% sucrose and
0.005% Tween 80.
[0185] Accordingly, a formulation of the invention may, in addition
to the polypeptide of the invention, comprise for example: [0186]
a) A buffer selected from a histidine buffer pH 6.5, histidine
buffer pH 6.0 and hepes buffer pH 7.0 at a concentration of 10 mM
to 100 mM; and [0187] b) An excipient selected from sucrose,
sorbitol, trehalose and mannitol at a concentration of 1% to
20%.
[0188] or [0189] a) A buffer selected from a histidine buffer pH
6.5, histidine buffer pH 6.0 and hepes buffer pH 7.0 at a
concentration of 10 mM to 100 mM; and [0190] c) A surfactant
selected from Tween 80, Tween 20 or a poloxamer at a concentration
of 0.001% to 1%.
[0191] or [0192] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 1% to 20%; and [0193]
c) A surfactant selected from Tween 80, Tween 20 or a poloxamer at
a concentration of 0.001% to 1%.
[0194] or [0195] a) A buffer selected from a histidine buffer pH
6.5, and hepes buffer pH 7.0 at a concentration of 15 mM; and
[0196] b) An excipient selected from sucrose, sorbitol, trehalose
and mannitol at a concentration of 1% to 20%.
[0197] or [0198] a) A buffer selected from a histidine buffer pH
6.5 and hepes buffer pH 7.0 at a concentration of 15 mM; and [0199]
c) A surfactant selected from Tween 80, Tween 20 or a poloxamer at
a concentration of 0.001% to 1%.
[0200] or [0201] a) A buffer selected from a histidine buffer pH
6.5 and hepes buffer pH 7.0 at a concentration of 10 mM to 100 mM;
and [0202] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 8%.
[0203] or [0204] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 8%; and [0205] c) A
surfactant selected from Tween 80, Tween 20 or a poloxamer at a
concentration of 0.001% to 1%.
[0206] or [0207] a) A buffer selected from a histidine buffer pH
6.5 and hepes buffer pH 7.0 at a concentration of 10 mM to 100 mM;
and [0208] c) A surfactant selected from Tween 80, Tween 20 or a
poloxamer at a concentration of 0.01%.
[0209] or [0210] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 1% to 20%; and [0211]
c) A surfactant selected from Tween 80, Tween 20 or a poloxamer at
a concentration of 0.01%.
[0212] or [0213] a) A buffer selected from a histidine buffer pH
6.0 at a concentration of 10 mM; and [0214] b) An excipient
selected from sucrose, sorbitol, trehalose and mannitol at a
concentration of 1% to 20%.
[0215] or [0216] a) A buffer selected from a histidine buffer pH
6.0 at a concentration of 10 mM; and [0217] c) A surfactant
selected from Tween 80, Tween 20 or a poloxamer at a concentration
of 0.001% to 1%.
[0218] or [0219] a) A buffer selected from a histidine buffer pH
6.0 at a concentration of 10 mM to 100 mM; and [0220] b) An
excipient selected from sucrose, sorbitol, trehalose and mannitol
at a concentration of 10%.
[0221] or [0222] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 10%; and [0223] c) A
surfactant selected from Tween 80, Tween 20 or a poloxamer at a
concentration of 0.001% to 1%.
[0224] or [0225] a) A buffer selected from a histidine buffer pH
6.0 at a concentration of 10 mM to 100 mM; and [0226] c) A
surfactant selected from Tween 80, Tween 20 or a poloxamer at a
concentration of 0.005%.
[0227] or [0228] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 1% to 20%; and [0229]
c) A surfactant selected from Tween 80, Tween 20 or a poloxamer at
a concentration of 0.005%.
[0230] or [0231] a) Histidine buffer pH 6.5 or pH 6.0 at a
concentration of 10 mM to 100 mM; and [0232] b) Sucrose at a
concentration of 1% to 20%.
[0233] or [0234] a) Histidine buffer pH 6.5 or pH 6.0 at a
concentration of 10 mM to 100 mM; and [0235] c) Tween 80 at a
concentration of 0.001% to 1%.
[0236] or [0237] b) Sucrose at a concentration of 1% to 20%; and
[0238] c) Tween 80 at a concentration of 0.001% to 1%.
[0239] or [0240] a) 15 mM histidine buffer pH 6.5; and [0241] b) 8%
sucrose.
[0242] or [0243] a) 15 mM histidine buffer pH 6.5; and [0244] c)
0.01% Tween 80.
[0245] or [0246] b) 8% sucrose; and [0247] c) 0.01% Tween 80.
[0248] or [0249] a) 10 mM histidine buffer pH 6.0; and [0250] b)
10% sucrose.
[0251] or [0252] a) 10 mM histidine buffer pH 6.0; and [0253] c)
0.005% Tween 80.
[0254] or [0255] b) 10% sucrose; and [0256] c) 0.005% Tween 80.
[0257] In a preferred aspect the formulation of the invention may
comprise a polypeptide selected from SEQ ID NO's: 1 to 6.
Accordingly the formulation of the invention may comprise:
[0258] a) 15 mM histidine buffer pH 6.5;
[0259] b) 8% sucrose; and
[0260] d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at
a concentration of 10 mg/ml),
[0261] or
[0262] a) 15 mM histidine buffer pH 6.5;
[0263] c) 0.01% Tween 80; and
[0264] d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at
a concentration of 10 mg/ml).
[0265] or
[0266] b) 8% sucrose;
[0267] c) 0.01% Tween 80; and
[0268] d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at
a concentration of 10 mg/ml).
[0269] or
[0270] a) 10 mM histidine buffer pH 6.0;
[0271] b) 10% sucrose; and
[0272] c) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at
a concentration of 10 mg/ml).
[0273] or
[0274] a) 10 mM histidine buffer pH 6.0;
[0275] c) 0.005% Tween 80; and
[0276] d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at
a concentration of 10 mg/ml).
[0277] or
[0278] b) 10% sucrose;
[0279] c) 0.005% Tween 80; and
[0280] d) A polypeptide selected from SEQ ID NO's: 1 to 6 (e.g. at
a concentration of 10 mg/ml).
[0281] In another preferred aspect, the formulation of the present
invention comprises an aqueous carrier having a pH of 5.5 to 8.0
and a polypeptide as defined above ("polypeptide of the invention")
comprising one or more single variable domains at a concentration
of 1 mg/mL to 200 mg/mL, said formulation being formulated for
administration to a human subject and comprises the components
selected from: [0282] a) A buffer at a concentration of 10 mM to
100 mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0; [0283] b) An excipient at a concentration of 1% to 20%;
and [0284] c) A surfactant at a concentration of 0.001% to 1%
selected from Tween 80, Tween 20 or a poloxamer; wherein said
formulation has an inorganic salt concentration of 150 mM or
lower.
[0285] Accordingly in this preferred aspect, in addition to the
polypeptide of the invention, the formulations of the present
invention may comprise at least an aqueous carrier having a pH of
5.5 to 8.0, a buffer selected from the group consisting of
histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH
6.0-6.5 and acetate pH 5.5-6.0, an excipient and a surfactant.
Preferably the buffer is selected from histidine pH 6.0-6.5 or
hepes pH 7.0. Most preferably the buffer is histidine pH 6.5 or
histidine pH 6.0. The buffer preferably has a concentration ranging
from 1 mM to 100 mM, 5 mM to 100 mM, 5 mM to 75 mM, 5 mM to 50 mM,
10 mM to 50 mM, 10 mM to 25 mM, 10 mM to 20 mM. In a specific
aspect, the concentration of buffer in the formulations of the
invention is 1 mM, 2 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 50 mM,
75 mM, or 100 mM. In a preferred aspect, the concentration is
between 10 and 20 mM, such as 10 mM or 15 mM. In a specific aspect,
a formulation of the invention comprises 15 mM histidine buffer pH
6.5. In another specific aspect, a formulation of the invention
comprises 10 mM histidine buffer pH 6.0. Preferred excipients are
polyols such as mannitol, sorbitol, etc., saccharides such as e.g.
sucrose, mannose, trehalose, etc. The concentration of the
excipient may range from about 1% to 20%, preferably from about
2.5% to 15%, more preferably from about 5% to 10%, such as e.g. 5%,
7.5%, 8% or 10%. In a preferred aspect, the formulation comprises
sucrose, preferably at a concentration of about 5% to 10%, such as
about 8% or 10%. The surfactant may be selected from Tween 20,
Tween 80 or a poloxamer. The concentration of the surfactant may
range from about 0.001% to 1% (preferably from about 0.001% to
0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%,
0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or
0.005%). In a specific embodiment, the surfactant is Tween 20 or
Tween 80, which is at a concentration of 0.001%, 0.005%, 0.01%,
0.02%, 0.05%, 0.08%, 0.1%, 0.5% or 1% of the formulation, such as
e.g. 0.01% Tween 80 or 0.005% Tween 80. Accordingly, a preferred
formulation of the invention may comprise 15 mM histidine pH 6.5,
8% sucrose and 0.01% Tween 80. Another preferred formulation of the
invention may comprise 10 mM histidine pH 6.0, 10% sucrose and
0.005% Tween 80.
[0286] Accordingly, a formulation of the invention may, in addition
to the polypeptide of the invention, comprise for example: [0287]
a) A buffer selected from a histidine buffer pH 6.5, a histidine
buffer pH 6.0 and hepes buffer pH 7.0 at a concentration of 10 mM
to 100 mM; [0288] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 1% to 20%; and [0289]
c) A surfactant selected from Tween 80, Tween 20 or a poloxamer at
a concentration of 0.001% to 1%. [0290] or [0291] a) A buffer
selected from a histidine buffer pH 6.5 and hepes buffer pH 7.0 at
a concentration of 15 mM; [0292] b) An excipient selected from
sucrose, sorbitol, trehalose and mannitol at a concentration of 1%
to 20%; and [0293] c) A surfactant selected from Tween 80, Tween 20
or a poloxamer at a concentration of 0.001% to 1%. [0294] or [0295]
a) A buffer selected from a histidine buffer pH 6.5 and hepes
buffer pH 7.0 at a concentration of 10 mM to 100 mM; [0296] b) An
excipient selected from sucrose, sorbitol, trehalose and mannitol
at a concentration of 8%; and [0297] c) A surfactant selected from
Tween 80, Tween 20 or a poloxamer at a concentration of 0.001% to
1%. [0298] or [0299] a) A buffer selected from a histidine buffer
pH 6.5 and hepes buffer pH 7.0 at a concentration of 10 mM to 100
mM; [0300] b) An excipient selected from sucrose, sorbitol,
trehalose and mannitol at a concentration of 1% to 20%; and [0301]
c) A surfactant selected from Tween 80, Tween 20 or a poloxamer at
a concentration of 0.01%. [0302] or [0303] a) A buffer selected
from a histidine buffer pH 6.5 and hepes buffer pH 7.0 at a
concentration of 15 mM; [0304] b) An excipient selected from
sucrose, sorbitol, trehalose and mannitol at a concentration of 8%;
and [0305] c) A surfactant selected from Tween 80, Tween 20 or a
poloxamer at a concentration of 0.001% to 1%. [0306] or [0307] a) A
buffer selected from a histidine buffer pH 6.5 and hepes buffer pH
7.0 at a concentration of 10 mM to 100 mM; [0308] b) An excipient
selected from sucrose, sorbitol, trehalose and mannitol at a
concentration of 8%; and [0309] c) A surfactant selected from Tween
80, Tween 20 or a poloxamer at a concentration of 0.01%. [0310] or
[0311] a) A buffer selected from a histidine buffer pH 6.5 and
hepes buffer pH 7.0 at a concentration of 15 mM; [0312] b) An
excipient selected from sucrose, sorbitol, trehalose and mannitol
at a concentration of 1% to 20%; and [0313] c) A surfactant
selected from Tween 80, Tween 20 or a poloxamer at a concentration
of 0.01%. [0314] or [0315] a) A buffer selected from a histidine
buffer pH 6.0 at a concentration of 10 mM; [0316] b) An excipient
selected from sucrose, sorbitol, trehalose and mannitol at a
concentration of 1% to 20%; and [0317] c) A surfactant selected
from Tween 80, Tween 20 or a poloxamer at a concentration of 0.001%
to 1%. [0318] or [0319] a) A buffer selected from a histidine
buffer pH 6.0 at a concentration of 10 mM to 100 mM; [0320] b) An
excipient selected from sucrose, sorbitol, trehalose and mannitol
at a concentration of 10%; and [0321] c) A surfactant selected from
Tween 80, Tween 20 or a poloxamer at a concentration of 0.001% to
1%. [0322] or [0323] a) A buffer selected from a histidine buffer
pH 6.0 at a concentration of 10 mM to 100 mM; [0324] b) An
excipient selected from sucrose, sorbitol, trehalose and mannitol
at a concentration of 1% to 20%; and [0325] c) A surfactant
selected from Tween 80, Tween 20 or a poloxamer at a concentration
of 0.005%. [0326] or [0327] a) A buffer selected from a histidine
buffer pH 6.0 at a concentration of 10 mM; [0328] b) An excipient
selected from sucrose, sorbitol, trehalose and mannitol at a
concentration of 10%; and [0329] c) A surfactant selected from
Tween 80, Tween 20 or a poloxamer at a concentration of 0.001% to
1%. [0330] or [0331] a) A buffer selected from a histidine buffer
pH 6.0 at a concentration of 10 mM to 100 mM; [0332] b) An
excipient selected from sucrose, sorbitol, trehalose and mannitol
at a concentration of 10%; and [0333] c) A surfactant selected from
Tween 80, Tween 20 or a poloxamer at a concentration of 0.005%.
[0334] or [0335] a) A buffer selected from a histidine buffer pH
6.0 at a concentration of 15 mM; [0336] b) An excipient selected
from sucrose, sorbitol, trehalose and mannitol at a concentration
of 1% to 20%; and [0337] c) A surfactant selected from Tween 80,
Tween 20 or a poloxamer at a concentration of 0.005%. [0338] or
[0339] a) Histidine buffer pH 6.5 at a concentration of 10 mM to
100 mM; [0340] b) Sucrose at a concentration of 1% to 20%; and
[0341] c) Tween 80 at a concentration of 0.001% to 1%. [0342] or
[0343] a) 15 mM histidine buffer pH 6.5; [0344] b) 8% sucrose; and
[0345] c) 0.01% Tween 80. [0346] or [0347] a) Histidine buffer pH
6.0 at a concentration of 10 mM to 100 mM; [0348] b) Sucrose at a
concentration of 1% to 20%; and [0349] c) Tween 80 at a
concentration of 0.001% to 1%. [0350] or [0351] a) 10 mM histidine
buffer pH 6.0; [0352] b) 10% sucrose; and [0353] c) 0.005% Tween
80.
[0354] In a preferred aspect the formulation of the invention may
comprise a polypeptide selected from SEQ ID NO's: 1 to 6.
Accordingly the formulation of the invention may comprise: [0355]
a) 15 mM histidine buffer pH 6.5; [0356] b) 8% sucrose; [0357] c)
0.01% Tween 80; and [0358] d) A polypeptide selected from SEQ ID
NO's: 1 to 6 (e.g. at a concentration of 10 mg/ml). [0359] or
[0360] a) 10 mM histidine buffer pH 6.0; [0361] b) 10% sucrose;
[0362] c) 0.005% Tween 80; and [0363] d) A polypeptide selected
from SEQ ID WO's: 1 to 6 (e.g. at a concentration of 10 mg/ml).
[0364] The components present in the formulations of the invention
have been selected such that the polypeptides of the invention have
a good solubility (as defined herein). Preferably, the polypeptides
present in the formulations of the present invention have a
solubility of at least 0.7 mM, at least 0.8 mM, at least 0.9 mM, at
least 1.0 mM, at least 1.1 mM, at least 1.2 mM, at least 1.3 mM, at
least 1.4 mM, at least 1.5 mM, at least 1.6 mM, at least 1.7 mM, at
least 1.8 mM, at least 1.9 mM, at least 2.0 mM, at least 2.1 mM, at
least 2.2 mM, at least 2.3 mM, at least 2.4 mM, at least 2.5 mM, at
least 2.6 mM, at least 2.7 mM, at least 2.8 mM, at least 2.9 mM, at
least 3.0 mM, at least 3.2 mM, at least 3.4 mM, at least 3.6 mM or
more and/or at least 20 mg/ml, at least 30 mg/mL, at least 40
mg/mL, at least 50 mg/mL, at least 60 mg/mL, at least 65 mg/mL, at
least 70 mg/mL, at least 80 mg/mL, at least 90 mg/mL, at least 100
mg/mL, at least 110 mg/mL, at least 120 mg/mL, at least 130 mg/mL,
at least 140 mg/mL, at least 150 mg/mL or even 200 mg/mL or more as
determined by the PEG exclusion method or by centrifugal
ultrafiltration. A very good solubility of the polypeptides of the
invention has been obtained with a formulation comprising a
histidine buffer pH 6.5 or with a formulation comprising Tween 80.
Accordingly, the present invention relates to a formulation
comprising an aqueous carrier and a polypeptide comprising one or
more single variable domains, said formulation being formulated for
administration to a human subject, wherein said formulation further
comprises at least one of: [0365] a) A histidine buffer pH 6.5 at a
concentration of 10 mM to 100 mM (preferably 10 mM to 50 mM, more
preferably 10 to 20 mM, such as 15 mM); [0366] c) Tween 80 at a
concentration of 0.001% to 1% (preferably from about 0.001% to
0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%,
0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01%);
wherein said formulation has an inorganic salt concentration of 150
mM or lower; and wherein the solubility of the polypeptide is at
least at least 20 mg/mL, at least 50 mg/mL, preferably at least 90
mg/mL, at least 120 mg/mL, at least 150 mg/mL or even 200 mg/mL or
more as determined by the PEG exclusion method or by centrifugal
ultrafiltration. In a preferred aspect, the formulation comprises a
histidine buffer pH 6.5 at a concentration of 10 mM to 100 mM
(preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such as 15
mM) and Tween 80 at a concentration of 0.001% to 1% (preferably
from about 0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%,
0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation,
preferably 0.01%).
[0367] Apart from this and/or in addition, the polypeptides of the
invention present in the formulation of the invention should
preferably have a melting temperature of at least 59.degree. C. or
more (such as 59.5.degree. C. or more), preferably at least
60.degree. C. or more (such as 60.5.degree. C. or more), more
preferably at least 61.degree. C. or more (such as 61.5.degree. C.
or more) or at least 62.degree. C. or more (such as 62.5.degree. C.
or more), most preferably at least 63.degree. C. or more (such as
63.5.degree. C. or more) as measured by the thermal shift assay
(TSA) and/or differential scanning calorimetry (DSC).
[0368] Without being limiting, melting point determination can be
done by the fluorescence-based thermal shift assay which is based
on the fact that upon thermal unfolding the hydrophobic regions of
proteins, usually hidden in the core of the protein fold, become
accessible for binding to a hydrophobic fluorescent dye. The
fluorescence emission of this dye is quenched in aqueous solution,
whereas upon binding to the hydrophobic patches of an unfolded
protein a sharp increase in the fluorescence yield of the probe is
observed. Temperature induced unfolding is typically a two-state
process with a sharp transition between the folded and unfolded
state, where the melting temperature (Tm) is defined as the
temperature at which half of the protein is in the unfolded state,
i.e. the first derivative of the fluorescence signal upon gradual
heating of the sample is plotted and the observed peak (or peaks
when multiple domains and/or variants of the same domain are
present) represents the melting temperature. The thermal shift
assay can be performed in a typical real-time PCR instrument where
melting curves can be recorded accurately in high-throughput mode
with only small quantities of protein required.
[0369] During a differential scanning calorimetry experiment the
sample is heated at a constant rate in an adiabatic environment
(.DELTA.T=0). The energy required to keep the temperature
difference between a reference and the sample cell at zero is
measured and yields the heat capacity as a function of temperature
(Cp(T)). The temperature corresponding to the maximum heat capacity
represents the melting temperature (T.sub.m). If the temperature
dependent unfolding process is reversible other thermodynamic
parameters such as the unfolding enthalpy (.DELTA.H.sub.unfolding)
can be determined.
[0370] Increased melting temperatures have been observed for the
polypeptides of the invention when present in a formulation with a
pH of about 6.0 to 8.0, preferably 6.2 to 7.5, more preferably 6.5
to 7.5, most preferably 6.5-7.0. Increased melting temperature have
also been obtained for the polypeptides of the invention when
present in a formulation that comprise hepes pH 7.0, histidine pH
6.0-6.5, MES pH 6.0 or acetate pH 6.0, or a formulation that
comprises an excipient, preferably a saccharides and/or polyol such
as mannitol, trehalose, sorbitol or sucrose. Accordingly, the
present invention relates to a formulation comprising an aqueous
carrier at a pH of 6.0 to 8.0 and a polypeptide comprising one or
more single variable domains, said formulation being formulated for
administration to a human subject, wherein said formulation further
comprises at least one of: [0371] a) A buffer at a concentration of
10 mM to 100 mM (preferably 10 mM to 50 mM, more preferably 10 to
20 mM, such as 15 mM or 10 mM) selected from the group consisting
of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH
6.0-6.5 and acetate pH 5.5-6.0; [0372] b) An excipient, preferably
a saccharide and/or polyol such as mannitol, sorbitol, trehalose or
sucrose at a concentration of 1% to 20% (preferably 2.5% to 15%,
more preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%). wherein
said formulation has an inorganic salt concentration of 150 mM or
lower; and wherein the melting temperature of the polypeptide of
the invention is at least 59.degree. C. or more (such as
59.5.degree. C. or more), preferably at least 60.degree. C. or more
(such as 60.5.degree. C. or more), more preferably at least
61.degree. C. or more (such as 61.5.degree. C. or more) or at least
62.degree. C. or more (such as 62.5.degree. C. or more), most
preferably at least 63.degree. C. or more (such as 63.5.degree. C.
or more) as measured by the thermal shift assay (TSA) and/or
differential scanning calorimetry (DSC).
[0373] In a preferred aspect, the formulation comprises a buffer at
a concentration of 10 mM to 100 mM (preferably 10 mM to 50 mM, more
preferably 10 to 20 mM, such as 15 mM) selected from the group
consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0,
succinate pH 6.0-6.5 and acetate pH 5.5-6.0, and an excipient,
preferably a saccharide and/or polyol such as mannitol, sorbitol,
trehalose or sucrose at a concentration of 1% to 20% (preferably
2.5% to 15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or
10%).
[0374] Apart from this and/or in addition, the formulation of the
present invention exhibits stability under at least one or more of
the following stress conditions: [0375] multiple (up to 10)
freeze/thaw cycles; [0376] storage at a temperature of 2-8.degree.
C. up to at least 2 weeks (preferably at least 3 weeks, at least 5
weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at
least 6 months, at least 1 year, 1.5 year or even 2 years or more);
[0377] storage at a temperature of 25.+-.5.degree. C. up to at
least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more); [0378]
storage at a temperature of 37.+-.5.degree. C. up to at least 2
weeks (preferably at least 3 weeks, at least 5 weeks, at least 8
weeks, at least 10 weeks, at least 3 months, at Least 6 months, at
least 1 year, 1.5 year or even 2 years or more); [0379] mechanical
stress.
[0380] Preferably the formulation of the invention is stable under
one or more of the following forms of mechanical stress: [0381]
shaking the formulation during 10 s to 1 min; [0382] pushing the
formulation through a needle (25 G, preferably 26 G, more
preferably 276, even more preferably 28 G, most preferably 296 or
more) with a syringe (the syringe used can be any commercially
available syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10
mL, 20 mL, 30 mL, 40 mL up to 50 mL syringe); [0383] rotating for
two days at 10 rpm; and/or [0384] stirring for 1 hour at room
temperature and/or 4-48 hours at 4.degree. C. at at least 10 rpm
(such as 50 rpm, 100 rpm or more).
[0385] Preferably, the formulation of the present invention is
stable under more than one of the above stress conditions, such as
at least two, at least three, at least four, at least five, at
least six, at least seven or most preferably under all of the above
stress conditions.
[0386] In one aspect, the formulation of the invention exhibits
stability under one or more forms of mechanical and/or shear
stress. Mechanical stress as used in the present invention can be
any form of external force applied on the formulation that may
affect the stability of the polypeptide present in the formulation.
Without being limiting, the mechanical stress applied to the
solution include shear stress, stir stress, shake stress, rotation
stress, etc. The formulation of the invention may for example be
shaken during at least 10 s, 20 s, 30 s, 40 s, 50 s up to 1 minute
or more. The formulation of the invention may be pushed through a
syringe (the syringe used can be any commercially available
syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL,
30 mL, 40 mL up to 50 mL syringe) with needle once, twice, three
times, four times, five times up to 10 times or more. Preferably
the needle has a size of 25 G (such as 26 G, 27 G, 28 G, 29 G, 30
G) or more. More preferably the size of the needles is 27 G or
more. In addition or alternatively, the formulation of the
invention may be rotated for 1 hour, 2 hours, 6 hours, 12 hours, 1
day up to two days or more at 10 rpm. The formulation of the
invention may be stirred for 1 hour at room temperature and/or 4
hours, 8 hours, 12 hours, 24 hours or even 48 hours or more at
2-8.degree. C. The speed of rotation is preferably above 10 rpm,
such as e.g. 50 rpm, 100 rpm or more.
[0387] The stability of the formulation under mechanical stress can
be assessed e.g. by visual inspection of the formulation or by
measurement of the OD320/OD280 ratio. Preferably the OD320/OD280
ratio is 0.05 or lower, such as 0.01 or 0.005.
[0388] A good stability of the polypeptides of the invention under
mechanical stress has been obtained with a formulation comprising
an excipient, preferably a saccharide and/or polyol such as
mannitol or sucrose or comprising Tween 80. Accordingly, the
present invention relates to a formulation comprising an aqueous
carrier and a polypeptide comprising one or more single variable
domains, said formulation being formulated for administration to a
human subject, wherein said formulation further comprises at least
one of: [0389] b) An excipient, preferably a saccharide and/or
polyol such as mannitol or sucrose at a concentration of 1% to 20%
(preferably 2.5% to 10%, more preferably 5% to 10%, such as 5%,
7.5%, 8% or 10%); [0390] c) A surfactant at a concentration of
0.001% to 1% (preferably from about 0.001% to 0.1%, or 0.01% to
0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%,
0.5%, or 1% of the formulation, preferably 0.01%) selected from
Tween 20, Tween 80 or a poloxamer, wherein said formulation has an
inorganic salt concentration of 150 mM or lower; and wherein the
polypeptide is stable under mechanical stress as determined by
OD320/OD280 ratio measurement. In a preferred aspect, the
formulation comprises an excipient, preferably a saccharide and/or
polyol such as mannitol or sucrose at a concentration of 1% to 20%
(preferably 2.5% to 15%, more preferably 5% to 10%, such as 5%,
7.5%, 8% or 10%) and a surfactant at a concentration of 0.001% to
1% (preferably about 0.001% to 0.1%, or 0.01% to 0.1% such as
0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of
the formulation, preferably 0.01% or 0.005%) selected from Tween 20
and Tween 80. A preferred formulation comprises 8% sucrose and
0.01% Tween 80, Another preferred formulation comprises 10% sucrose
and 0.005% Tween 80.
[0391] In a specific aspect, the formulation of the invention
comprises an excipient, preferably a saccharide and/or polyol such
as mannitol or sucrose at a concentration of 1% to 20% (preferably
2.5% to 15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or
10%) and/or Tween 20, Tween 80 or a poloxamer (at a concentration
ranging from about 0.001% to 0.1%, or 0.01% to 0.1% such as 0.001%,
0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the
formulation, preferably 0.01%) and is characterized that the
polypeptides present in the formulation are stable under mechanical
stress.
[0392] In a preferred aspect, the formulation of the invention
comprises an excipient, preferably a saccharide and/or polyol such
as mannitol or sucrose at a concentration of 1% to 20% (preferably
2.5% to 15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or
10%) and/or Tween 80 (at a concentration of 0.001%, 0.005%, 0.01%,
0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation,
preferably 0.01% or 0.005%) and is characterized that the
polypeptides present in the formulation of the invention are stable
when shaking the formulation during at least 10 s, 20 s, 30 s, 40
s, 50 s up to 1 minute of more. In another preferred aspect, the
formulation of the invention comprises an excipient, preferably a
saccharide and/or polyol such as mannitol or sucrose at a
concentration of 1% to 20% (preferably 2.5% to 15%, more preferably
5% to 10%, such as 5%, 7.5%, 8% or 10%) and/or Tween 80 (at a
concentration of 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%,
0.5%, or 1% of the formulation, preferably 0.01% or 0.005%) and is
characterized that the polypeptides present in the formulation of
the invention are stable when pushing the formulation through a
syringe (the syringe used can be any commercially available
syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL,
30 mL, 40 mL up to 50 mL syringe) with needle size of 25 G or more
(such as 26 G, 27 G, 28 G, 29 G, 30 G or more, preferably 27 G or
more) once, twice, three times, four times, five times up to 10
times or more. In another preferred aspect, the formulation of the
invention comprises an excipient, preferably a saccharide and/or
polyol such as mannitol or sucrose at a concentration of 1% to 20%
(preferably 2.5% to 15%, more preferably 5% to 10%, such as 5%,
73%, 8% or 10%) and/or Tween 80 (at a concentration of 0.001%,
0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the
formulation, preferably 0.01% or 0.005%) and is characterized that
the polypeptides present in the formulation of the invention are
stable when rotating the formulation for 1 hour, 2 hours, 6 hours,
12 hours, 1 day up to two days or more at 10 rpm. In another
preferred aspect, the formulation of the invention comprises an
excipient, preferably a saccharide and/or polyol such as mannitol
or sucrose at a concentration of 1% to 20% (preferably 2.5% to 15%,
more preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%) and/or
Tween 80 (at a concentration of 0.001%, 0.005%, 0.01%, 0.02%,
0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably
0.01% or 0.005%) and is characterized that the polypeptides present
in the formulation of the invention are stable when stirring the
formulation for 1 hour at room temperature and/or 4 hours, 8 hours,
12 hours, 24 hours or even 48 hours or more at 2-8.degree. C. at at
least 10 rpm (such as 50 rpm, 100 rpm or more).
[0393] A "freeze/thaw cycle" or "F/T cycle" is defined as the
freezing of a sample in a freezer (-20.+-.5.degree. C.) or
ultrafreezer (below -64.degree. C. (such as e.g. at -80.degree.
C.)) until solid, followed by thawing at room temperature until all
ice crystals have visually disappeared. In one aspect, the
formulation of the invention exhibits stability under multiple (up
to 10) freeze/thaw cycles. The formulation of the invention may
exhibit stability under at least 1, at least 2, at least 3, at
least 5 to up to at least 10 freeze/thaw cycles, such as e.g. 10
cycles at -20.degree. C., 2 cycles at -80.degree. C.+1 cycle at
-20.degree. C. or 2 cycles at -80.degree. C.+6 cycles at
-20.degree. C.
[0394] In yet another aspect, the formulation of the invention
exhibits stability when stored at a temperature of 5.+-.5.degree.
C. The formulation of the invention may exhibit stability when
stored at a temperature of 5.+-.5.degree. C. for at least 2 weeks,
3 weeks, 4 weeks, 8 weeks, 10 weeks, up to 3 months, 6 months, 11
months, 1 year, 1.5 year or even 2 years and more.
[0395] In yet another aspect, the formulation of the invention
exhibits stability when stored at a temperature of 25.+-.5.degree.
C. The formulation of the invention may exhibit stability when
stored at a temperature of 25.+-.5.degree. C. for at least 2 weeks,
3 weeks, 4 weeks, at least 5 weeks, at least 8 weeks, at least 10
weeks, at least 3 months, at least 6 months, at least 1 year, 1.5
year or even 2 years or more.
[0396] In yet another aspect, the formulation of the invention
exhibits stability when stored at a temperature of 37.+-.5.degree.
C. The formulation of the invention may exhibit stability when
stored at a temperature of 37.+-.5.degree. C. for at least 2 weeks,
3 weeks, 4 weeks, at least 5 weeks, at least 8 weeks, at least 10
weeks, at least 3 months, at least 6 months, at least 1 year, 1.5
year or even 2 years or more.
[0397] As is known to one skilled in the art, the temperatures
indicated in this text can be subject to normal variations.
[0398] Preferably, in those formulations that are stable under one
or more of the above stress conditions: [0399] less than 10% (more
preferably less than 5%, even more preferably less than 3%, most
preferably less than 1%) of the polypeptide of the invention forms
pyroglutamate at the N-terminal glutamic acid (e.g. as assessed by
RP-HPLC) during storage under (one of the above) stress conditions,
such as e.g. at a temperature of 37.+-.5.degree. C. up to at least
2 weeks (preferably at least 3 weeks, at least 5 weeks, at least 8
weeks, at least 10 weeks, at least 3 months, at least 6 months, at
least 1 year, 1.5 year or even 2 years or more); [0400] less than
10% (more preferably less than 5%, even more preferably less than
3%, most preferably less than 1%) of the polypeptide of the
invention forms dimers (e.g. as assessed by SE-HPLC) during storage
under (one of the above) stress conditions, such as e.g. at a
temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more); [0401] at least 80% (at
least 85%, at least 90%, at least 95%, at least 98%, at least 99%,
or at least 99.5%) of the polypeptides of the invention retain
their binding activity (e.g. as assessed by ELISA and/or Biacore)
to at least one of their (preferably to all of their) targets after
storage under (one of the above) stress conditions, such as e.g. at
a temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more) compared to the binding
activity prior to the stress condition.
[0402] Stability of the formulations can be assessed by various
analytical and/or immunological methods known in the art. The
protein content of the polypeptides of the invention can, for
example be detected by spectrophotometrical methods.
[0403] SDS-PAGE allows the visualization of the polypeptides in a
given sample via direct staining. SDS-PAGE is used to separate
proteins according to their size. Both reducing and non-reducing
SDS-PAGE analysis can be performed.
[0404] The molecular size distribution and the relative amounts of
polypeptide of the invention and protein impurities can be
determined by Size Exclusion High Performance Liquid Chromatography
(SE-HPLC). The relative amount of a specific protein impurity,
expressed as area %, can be calculated by dividing the peak area
corresponding to the impurity by the total integrated area. SE-HPLC
methods are known to the skilled person and are also described in
the Example section.
[0405] Reversed Phase High Performance Liquid chromatography
(RP-HPLC) separates molecules with respect to differences in
hydrophobicity and is based on the reversible interaction between
the molecule and the hydrophobic stationary phase. In this assay a
Zorbax 300SB-C3 column (Agilent Technologies, Palo Alto, US) can be
used. The relative amount of a specific protein impurity, expressed
as area %, can be calculated by dividing the peak area
corresponding to the impurity by the total integrated area. RP-HPLC
methods are known to the skilled person and are also described in
the Example section.
[0406] Polypeptides of the invention and their charge variants can
be separated by Ion Exchange
[0407] High Performance Liquid Chromatography (IEX-HPLC). Also
potential impurities can be detected with this method. The relative
amount of a specific protein impurity, expressed as area %, can be
calculated by dividing the peak area corresponding to the impurity
by the total integrated area. IEX-HPLC methods are known to the
skilled person and are also described in the Example section.
[0408] The polypeptides present in the formulation of the invention
preferably do not form pyroglutamate at the N-terminal glutamic
acid. The formation of pyroglutamate in the sample can e.g. be
measured by RP-HPLC. For example, analysis by RP-HPLC of a
formulation containing SEQ ID NO: 4 after storage for 10 weeks at a
temperature of 37.degree. C., showed the formation of pyroglutamate
as a separate peak at 18-19 minutes. Preferably in the formulation
of the invention, less than 10% (more preferably less than 5%, even
more preferably less than 3%, most preferably less than 1%) of the
polypeptides form pyroglutamate at the N-terminal glutamic acid
(e.g. as assessed by RP-HPLC) under one or more of the above stress
conditions.
[0409] Little to no pyroglutamate formation of the polypeptides of
the invention has been observed in formulations with a pH of 7.0 or
lower, preferably 6.5 or lower, such as 6.5, 6.0 or 5.5, in e.g.
histidine buffers and acetate buffers. Accordingly, the present
invention relates to a formulation comprising an aqueous carrier at
a pH of 7.0 or lower and a polypeptide comprising one or more
single variable domains, said formulation being formulated for
administration to a human subject, wherein said formulation further
comprises: [0410] a) A buffer at a concentration of 10 mM to 100 mM
(preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such as 15
mM or 10 mM) selected from the group consisting of histidine pH
6.0-6.5 and acetate pH 5.5-6.0, wherein said formulation has an
inorganic salt concentration of 150 mM or lower; and wherein less
than 10% (preferably less than 8%, more preferably less than 7%,
most preferably less than 5%) of the polypeptides forms
pyroglutamate at the N-terminal glutamic acid during one or more of
the above stress conditions (such as during storage at a
temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 5 months, at least 1
year, 1.5 year or even 2 years or more)), the % of pyroglutamate as
measured by RP-HPLC. In a preferred aspect, the formulation
comprises a histidine buffer pH 6.5 at 15 mM. In another preferred
aspect, the formulation comprises a histidine buffer pH 6.0 at 10
mM.
[0411] The polypeptides present in the formulation of the invention
also preferably do not form dimers. The formation of dimers in the
sample can e.g. be measured by SE-HPLC. For example, analysis in
SE-HPLC of a formulation containing SEQ ID NO: 4 after storage for
10 weeks at a temperature of 37.degree. C., showed the formation of
a separate peak eluting at an apparent molecular weight of 44 kDa
in comparison with molecular weight markers, while the monomeric
polypeptide eluted between the 44 and 17 kDa molecular weight
markers. This separate peak at 44 kDa represented a dimeric form of
SEQ ID NO: 4. Preferably in the formulation of the invention, less
than 10% (more preferably less than 5%, even more preferably less
than 3%, most preferably less than 1%) of the polypeptides forms
dimers (e.g. as assessed by SE-HPLC) during storage under one or
more of the above stress conditions.
[0412] Little to no dimer formation of the polypeptides of the
invention has been observed in formulations with a histidine buffer
or an acetate buffer and in formulations that comprise an
excipient, preferably a saccharide and/or polyol such as mannitol,
trehalose, sorbitol or sucrose. Accordingly, the present invention
relates to a formulation comprising an aqueous carrier and a
polypeptide comprising one or more single variable domains, said
formulation being formulated for administration to a human subject,
wherein said formulation further comprises at least one of: [0413]
a) A buffer at a concentration of 10 mM to 100 mM (preferably 10 mM
to 50 mM, more preferably 10 to 20 mM, such as 15 mM or 10 mM)
selected from the group consisting of histidine pH 6.0-6.5 and
acetate pH 5.5-6.0; [0414] b) An excipient, preferably a
saccharide, a non-reducing sugar and/or polyol such as mannitol,
trehalose, sorbitol or sucrose at a concentration of 1% to 20%
(preferably 2.5% to 15%, more preferably 5% to 10%, such as 5%,
7.5%, 8% or 10%), wherein said formulation has an inorganic salt
concentration of 150 mM or lower; and wherein less than 10%
(preferably less than 8%, more preferably less than 7%, most
preferably less than 5%) of the polypeptides forms dimers during
one or more of the above stress conditions (such as during storage
at a temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more)), the % of dimers as
measured by SE-HPLC. In a preferred aspect, the formulation
comprises a histidine buffer pH 6.5 at 15 mM. In another preferred
aspect, the formulation comprises a histidine buffer pH 6.0 at 10
mM. In a preferred aspect, the formulation comprises a buffer at a
concentration of 10 mM to 100 mM (preferably 10 mM to 50 mM, more
preferably 10 to 20 mM, such as 15 mM or 10 mM) selected from the
group consisting of histidine pH 6.0-6.5 and acetate pH 5.5-6.0 and
an excipient, preferably a saccharide, non-reducing sugar and/or
polyol such as mannitol, trehalose, sorbitol or sucrose at a
concentration of 1% to 20% (preferably 2.5% to 15%, more preferably
5% to 10%, such as 5%, 7.5%, 8% or 10%), such as e.g. 15 mM
histidine pH 6.5 and 8% sucrose; or 10 mM histidine pH 6.0 and 10%
sucrose.
[0415] Preferably in the formulation of the invention, less than
10% (more preferably less than 5%, even more preferably less than
3%, most preferably less than 1%) of the polypeptides forms
pyroglutamate at the N-terminal glutamic acid (e.g. as assessed by
RP-HPLC) and less than 10% (more preferably less than 5%, even more
preferably less than 3%, most preferably less than 1%) of the
polypeptides forms dimers (e.g. as assessed by SE-HPLC) during
storage under one or more of the above stress conditions.
[0416] Apart from this and/or in addition, the formulation of the
present invention should show only low to undetectable levels of
aggregation even during storage under one or more of the above
stress conditions. For example, in the formulation of the
invention, no more than 5%, no more than 4%, no more than 3%, no
more than 2%, no more than 1%, and most preferably no more than
0.5% of the polypeptides forms an aggregate after storage under one
or more of the above stress conditions.
[0417] Aggregation as used in the present invention means the
development of high molecular weight aggregates, i.e. aggregates
with an apparent molecular weight in SE-HPLC analysis of
more/higher than the molecular weight observed for dimers. As
described above, 44 kDa is the apparent molecular weight observed
in SE-HPLC analysis for dimers of SEQ ID NO: 4, 36-38 kDa is the
apparent molecular weight observed in SE-HPLC analysis for dimers
of SEQ ID NO's: 1-3 and 36 kDa is the apparent molecular weight
observed in SE-HPLC analysis for dimers of SEQ ID NO: 5.
Aggregation can be assessed by various methods known in the art.
Without being limiting, examples include SE-HPLC, analytical
ultracentrifugation, dynamic light scattering, subvisible particle
counting and OD320/OD280 measurement.
[0418] In an analytical ultracentrifuge, a sample being spun can be
monitored in real time through an optical detection system, using
ultraviolet light absorption and/or interference optical refractive
index sensitive system. This allows the operator to observe the
evolution of the sample concentration versus the axis of rotation
profile as a result of the applied centrifugal field. With modern
instrumentation, these observations are electronically digitized
and stored for further mathematical analysis. Two kinds of
experiments are commonly performed on these instruments:
sedimentation velocity experiments and sedimentation equilibrium
experiments.
[0419] Sedimentation velocity experiments aim to interpret the
entire time-course of sedimentation, and report on the shape and
molar mass of the dissolved macromolecules, as well as their
size-distribution (Perez-Ramirez and Steckert (2005) Therapeutic
Proteins: Methods and Protocols. C. M. Smales and D. C. James, Eds.
Vol. 308: 301-318. Humana Press Inc Totowa, N.J., US.). The size
resolution of this method scales approximately with the square of
the particle radii, and by adjusting the rotor speed of the
experiment size-ranges from 100 Da to 10 GDa can be covered.
Sedimentation velocity experiments can also be used to study
reversible chemical equilibria between macromolecular species, by
either monitoring the number and molar mass of macromolecular
complexes, by gaining information about the complex composition
from multi-signal analysis exploiting differences in each
components spectroscopic signal, or by following the composition
dependence of the sedimentation rates of the macromolecular system,
as described in Gilbert-Jenkins theory.
[0420] Sedimentation equilibrium experiments are concerned only
with the final steady-state of the experiment, where sedimentation
is balanced by diffusion opposing the concentration gradients,
resulting in a time-independent concentration profile.
Sedimentation equilibrium distributions in the centrifugal field
are characterized by Boltzmann distributions. This experiment is
insensitive to the shape of the macromolecule, and directly reports
on the molar mass of the macromolecules and, for chemically
reacting mixtures, on chemical equilibrium constants.
[0421] The kinds of information that can be obtained from an
analytical ultracentrifuge include the gross shape of
macromolecules, the conformational changes in macromolecules, and
size distributions of macromolecular samples. For macromolecules,
such as proteins, that exist in chemical equilibrium with different
non-covalent complexes, the number and subunit stoichiometry of the
complexes and equilibrium constant constants can be studied. (see
also Scott D. I., Harding S. E. and Rowe A. J. Analytical
Ultracentrifugation Techniques and Methods, RSC Publishing)
[0422] Dynamic light scattering (also known as Photon Correlation
Spectroscopy or quasi-elastic light scattering) is a technique in
physics, which can be used to determine the size distribution
profile of small particles in solution. When a beam of light passes
through a colloidal dispersion, the particles or droplets scatter
some of the light in all directions. When the particles are very
small compared with the wavelength of the light, the intensity of
the scattered light is uniform in all directions (Rayleigh
scattering); for larger particles (above approximately 250 nm
diameter), the intensity is angle dependent (Mie scattering). If
the light is coherent and monochromatic, as from a laser for
example, it is possible to observe time-dependent fluctuations in
the scattered intensity using a suitable detector such as a
photomultiplier capable of operating in photon counting mode.
[0423] These fluctuations arise from the fact that the particles
are small enough to undergo random thermal (Brownian) motion and
the distance between them is therefore constantly varying.
Constructive and destructive interference of light scattered by
neighbouring particles within the illuminated zone gives rise to
the intensity fluctuation at the detector plane which, as it arises
from particle motion, contains information about this motion.
Analysis of the time dependence of the intensity fluctuation can
therefore yield the diffusion coefficient of the particles from
which, via the Stokes Einstein equation, knowing the viscosity of
the medium, the hydrodynamic radius or diameter of the particles
can be calculated. (see also Berne B. J. and Pecora R. Dynamic
Light Scattering With Applications to Chemistry, Biology and
Physics, Dover Publications)
[0424] Aggregation can also be measured by the PAMAS SVSS-C (Small
Volume Syringe System-C) instrument (PArtikelMess and
AnalyseSysteme GMBH), which is a particle size distribution
analyzer for low viscous fluids. It uses the principle of light
obscuration to detect sub-visible particles in the size range 1
.mu.m-200 .mu.m. The validation criteria/specified limits of the
European Pharmacopoeia (EP<2.9.19 Particulate Contamination:
sub-visible particles) for small and large volume parenterals are
defined by the total counts per container:
[0425] For particles >10 .mu.m, no more than 6000 counts per
container
[0426] For particles >25 .mu.m, no more than 600 counts per
container
[0427] The OD320/OD280 ratio is also a measure for turbidity or the
presence of particulates in the sample. In a preferred aspect, the
OD320/OD280 ratio of the formulation of the invention should be
0.05 or lower, preferably 0.01 or lower, such as 0.005 or
lower.
[0428] The tendency for aggregate formation of a polypeptide in a
certain formulation can also be measured by elastic light
scattering. Elastic light scattering can be measured in a
spectrofluorometer (e.g. excitation and emission wavelength 500 nm)
by temperature-induced denaturation as measured e.g. at an angle of
90.degree.. Preferably the maximum scatter will stay within the
absorption detection limit. The scatter should be 1000 abs. or
lower, preferably 750 abs or lower, such as 500 abs or lower.
[0429] No particulate formation has been observed in formulations
comprising a histidine buffer pH 6.0-6.5 under different stress
conditions (such as e.g. storage at a temperature of 5.+-.5.degree.
C. up to up to at least 2 weeks (preferably at least 3 weeks, at
least 5 weeks, at least 8 weeks, at least 10 weeks, at least 3
months, at least 6 months, at least 1 year, 1.5 year or even 2
years or more) or storage at a temperature of 37.+-.5.degree. C. up
to at least 2 weeks (preferably at least 3 weeks, at least 5 weeks,
at least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more)).
Accordingly, the present invention relates to a formulation
comprising an aqueous carrier and a polypeptide comprising one or
more single variable domains, said formulation being formulated for
administration to a human subject, wherein said formulation further
comprises: [0430] a) A histidine buffer pH 6.0-6.5 at a
concentration of 10 mM to 100 mM (preferably 10 mM to 50 mM, more
preferably 10 to 20 mM, such as 15 mM), wherein said formulation
has an inorganic salt concentration of 150 mM or lower; and wherein
no particulates are present under one or more of the above stress
conditions (e.g. when stored at a temperature of 5.+-.5.degree. C.
up to up to at least 2 weeks (preferably at least 3 weeks, at least
5 weeks, at least 8 weeks, at least 10 weeks, at least 3 months, at
least 6 months, at least 1 year, 1.5 year or even 2 years or more)
or when stored at a temperature of 37.+-.5.degree. C. up to at
least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more)) as
measured by OD320/OD280 ratio measurement and/or elastic light
scattering. In a preferred aspect the formulation comprises a
histidine buffer pH 6.5 at 15 mM. In another preferred aspect the
formulation comprises a histidine buffer pH 6.0 at 10 mM.
[0431] Apart from this and/or in addition, the formulation of the
present invention shows only low to undetectable levels of
fragmentation and/or degradation even during storage under one or
more of the above stress conditions. Fragmentation and degradation
can be measured e.g. by SE-HPLC and/or RP-HPLC. For example,
analysis in SE-HPLC of a formulation containing SEQ ID NO: 4 after
storage for 10 weeks at a temperature of 37.degree. C., showed the
formation of some minor postpeaks, representing degradation
products of SEQ ID NO: 4. For example, analysis by RP-HPLC of a
formulation containing SEQ ID NO: 4 after storage for 10 weeks at a
temperature of 37.degree. C., showed the formation of some minor
peaks at 8-9 minutes, representing degradation products. Preferably
in the formulation of the invention, no more than 5%, no more than
4%, no more than 3%, no more than 2%, no more than 1%, no more than
0.5%, no more than 0.1%, no more than 0.05%, and most preferably no
more than 0.01% of the polypeptides shows degradation and/or
fragmentation after storage under one or more of the above stress
conditions.
[0432] The techniques of static light scattering (SLS), tangential
flow filtration (TFF), Fourier Transform Infrared Spectroscopy
(FTIR), circular dichroism (CD), urea-induced protein unfolding
techniques, intrinsic tryptophan fluorescence and/or
1-anilino-8-naphthalenesulfonic acid (ANS) protein binding can also
be used to assess the physical properties and stability of
polypeptides.
[0433] Apart from this and/or in addition, the formulation of the
present invention shows very little to no loss of potency and/or
biological activity of their polypeptides, even during storage
under one or more of the above stress conditions.
[0434] The potency and/or biological activity of a biological
describes the specific ability or capacity of said biological to
achieve a defined biological effect. The potency and biological
activities of the polypeptides of the invention can be assessed by
various assays including any suitable in vitro assay, cell-based
assay, in vivo assay and/or animal model known per se, or any
combination thereof, depending on the specific disease or disorder
involved. Suitable in vitro assays will be clear to the skilled
person, and for example include ELISA; FACS binding assay; Biacore;
competition binding assay (AlphaScreen.RTM., Perkin Elmer, Mass.,
USA; FMAT); TRAP assay (osteoclast differentiation assay; Rissanen
et al. 2005, J. Bone Miner. Res. 20, Suppl. 1: S256); NF-kappaB
reporter gene assay (Mizukami et al. 2002, Mol. Cell. Biol. 22:
992-1000). For example, SEQ ID NO: 4 interacts with RANKL and
blocks the interaction of this ligand with RANK, thereby preventing
signalization through this receptor. SEQ ID NO's: 1 to 3 interact
with IL-6R and block the interaction of this receptor with IL-6.
SEQ ID NO's: 5 and 6 interact with IL-23 and block the interaction
of this ligand with its receptor. The potency of SEQ ID NO's: 1 to
6 for blocking the respective ligand/receptor interaction can be
determined, e.g. by ELISA, Biacore, AlphaScreen.RTM..
[0435] For example, in one embodiment, Biacore kinetic analysis
uses Surface Plasmon Resonance (SPR) technology to monitor
macromolecular interactions in real time and is used to determine
the binding on and off rates of polypeptides of the formulation of
the invention to their target. Biacore kinetic analysis comprises
analyzing the binding and dissociation of the target from chips
with immobilized polypeptides of the invention on their surface. A
typical Biacore kinetic study involves the injection of 250 .mu.L
of polypeptide reagent at varying concentration in HBS buffer
containing 0.005% Tween 20 over a sensor chip surface, onto which
has been immobilized the antigen. In the BIAcore 3000 system, the
ligand is immobilized on carboxymethylated dextran over a gold
surface, while the second partner (analyte) is captured as it flows
over the immobilized ligand surface. The immobilized ligands are
remarkably resilient and maintain their biological activity. The
bound analytes can be stripped from the immobilized ligand without
affecting its activity to allow many cycles of binding and
regeneration on the same immobilized surface. Interaction is
detected in real time via SPR and at high sensitivity. Because the
same affinity may reflect different on-rates and off-rates, this
instrument excels over most other affinity measuring methods in
that it measures on-rates (ka) and off-rates (kd). Concentration
determination experiments are also feasible.
[0436] The formulation of the present invention exhibits almost no
loss in biological activities of the polypeptide during the
prolonged storage under the conditions described above, as assessed
by various immunological assays including, for example,
enzyme-linked immunosorbent assay (ELISA) and Surface Plasmon
Resonance to measure the ability of the polypeptide to specifically
bind to an antigen. The polypeptides present in the formulation of
the present invention retain, even under the above defined stress
conditions (such as storage under certain temperature stress for
defined periods) more than 80%, more than 85%, more than 90%, more
than 95%, more than 98%, more than 99%, or more than 99.5% of their
initial biological activities (e.g., the ability to bind to RANKL,
IL-6R, IL-23 and/or HSA) of the polypeptides prior to the storage.
In some embodiments, the polypeptides in the formulation of the
invention retain under the above defined stress conditions at least
80%, at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or at least 99.5% of the biological activity (e.g., the
ability to bind to RANKL, IL-6R, IL-23 and/or HSA) compared to the
polypeptides present in a reference formulation prior to the
storage.
[0437] In one embodiment, the polypeptides of the invention binds
HSA. In the formulations of the present invention, at least 80%
(preferably at least 85%, at least 90%, at least 95%, at least 98%,
at least 99%, or at least 99.5%) of said polypeptides retain their
binding activity to NSA under one or more of the above stress
conditions (such as storage under certain temperature stress for
defined periods) compared to the binding activity prior to the
stress condition. Without being limiting, the binding of the
polypeptides to HSA can be determined e.g. by ELISA and/or
Biacore.
[0438] In another embodiment, the polypeptides of the invention
bind RANKL. In the formulation of the present invention at least
80% (at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or at least 99.5%) of said polypeptides retain their
binding activity to RANKL after storage under one or more of the
above stress conditions compared to the binding activity prior to
storage.
[0439] In another embodiment, the polypeptides of the invention
bind IL-6R. In the formulation of the present invention at least
80% (at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or at least 99.5%) of said polypeptides retain their
binding activity to IL-6R after storage under one or more of the
above stress conditions compared to the binding activity prior to
storage.
[0440] In another embodiment, the polypeptides of the invention
bind IL-23. In the formulation of the present invention at least
80% (at least 85%, at least 90%, at least 95%, at least 98%, at
least 99%, or at least 99.5%) of said polypeptides retain their
binding activity to IL-23 after storage under one or more of the
above stress conditions compared to the binding activity prior to
storage.
[0441] in a preferred aspect, at least 80% (at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)
of the polypeptides present in the formulation of the invention
retain their binding activity to all of their targets (such as e.g.
RANKL and HSA, IL-6R and NSA or IL-23 and NSA) after storage under
one or more of the above stress conditions compared to the binding
activity prior to storage.
[0442] Suitable animal models for determining the potency and/or
biological activity of the polypeptides present in the formulations
of the invention will be clear to the skilled person and will
depend on the intended disease and/or disorder to be prevented
and/or treated by the polypeptide of the invention. Suitable animal
models for testing the potency and/or biological activity of SEQ ID
NO's: 1 to 6 are e.g. described in WO 08/020,079, WO 09/068,627 and
WO 08/142,164.
[0443] Little to no loss of potency of the polypeptides of the
invention has been observed in formulations with a histidine buffer
and in formulations that comprise an excipient, preferably a
saccharide, non-reducing sugar and/or polyol such as mannitol,
sorbitol, trehalose or sucrose. Accordingly, the present invention
relates to a formulation comprising an aqueous carrier and a
polypeptide comprising one or more single variable domains, said
formulation being formulated for administration to a human subject,
wherein said formulation further comprises at least one of: [0444]
a) A histidine buffer at a concentration of 10 mM to 100 mM
(preferably 10 mM to 50 mM, more preferably 10 to 20 mM, such as 15
mM or 10 mM); [0445] b) An excipient, preferably a saccharide,
non-reducing sugar and/or polyol such as mannitol, sorbitol,
trehalose or sucrose at a concentration of 1% to 20% (preferably
2.5% to 15%, more preferably 5% to 10%, such as 5%, 7.5%, 8% or
10%), wherein said formulation has an inorganic salt concentration
of 150 mM or lower; and wherein at least 80% (preferably at least
85%, at least 90%, at least 95%, at least 98%, at least 99%, or at
least 99.5%) of the polypeptides retain their binding activity to
at least one (preferably to all) of their targets under one or more
of the above stress conditions (such as during storage at a
temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more)) compared to the binding
activity prior to the stress conditions, said binding activity as
measured by ELISA and/or Biacore. In a preferred aspect, the
formulation comprises a histidine buffer at a concentration of 10
mM to 100 mM (preferably 10 mM to 50 mM, more preferably 10 to 20
mM, such as 15 mM or 10 mM) and an excipient, preferably a
saccharide, non-reducing sugar and/or polyol such as mannitol,
sorbitol, trehalose or sucrose at a concentration of 1% to 20%
(preferably 2.5% to 15%, more preferably 5% to 10%, such as 5%,
7.5%, 8% or 10%), such as e.g. 15 mM histidine pH 6.5 and 8%
sucrose; or 10 mM histidine pH 6.0 and 10% sucrose.
[0446] Accordingly, in the stable formulations of the present
invention preferably: [0447] the polypeptide of the invention has a
solubility of at least 20 mg/mL, at least 50 mg/mL, preferably at
least 90 mg/mL, at least 120 mg/mL, at least 150 mg/mL or even 200
mg/mL or more) (e.g. as assessed by PEG exclusion method or by
centrifugal ultrafiltration); [0448] the polypeptide of the
invention has a melting temperature of at least 59.degree. C. or
more (such as 59.5.degree. C. or more), preferably at least
60.degree. C. or more (such as 60.5.degree. C. or more), more
preferably at least 61.degree. C. or more (such as 61.5.degree. C.
or more) or at least 62.degree. C. or more (such as 62.5.degree. C.
or more), most preferably at least 63.degree. C. or more (such as
63.5.degree. C. or more) (e.g. as assessed by TSA or DSC); [0449]
less than 10% (more preferably less than 5%, even more preferably
less than 3%, most preferably less than 1%) of the polypeptide of
the invention forms pyroglutamate at the N-terminal glutamic acid
(e.g. as assessed by RP-HPLC) during storage under one or more (of
the above) stress conditions, such as e.g. at a temperature of
37.+-.5.degree. C. up to at least 2 weeks (preferably at least 3
weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more); [0450] less than 10% (more preferably less
than 5%, even more preferably less than 3%, most preferably less
than 1%) of the polypeptide of the invention forms dimers (e.g. as
assessed by SE-HPLC) during storage under one or more (of the
above) stress conditions, such as e.g. at a temperature of
37.+-.5.degree. C. up to at least 2 weeks (preferably at least 3
weeks, at least 5 weeks, at least 8 weeks, at least 10 weeks, at
least 3 months, at least 6 months, at least 1 year, 1.5 year or
even 2 years or more); [0451] at least 80% (at least 85%, at least
90%, at least 95%, at least 98%, at least 99%, or at least 99.5%)
of the polypeptides of the invention retain their binding activity
(e.g. as assessed by ELISA and/or Biacore) to at least one
(preferably to all) of their targets after storage under one or
more (of the above) stress conditions, such as e.g. at a
temperature of 37.+-.-5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more) compared to the binding
activity prior to the stress condition; and/or [0452] the
polypeptide of the invention is stable under one or more of the
following mechanical stress conditions: [0453] shaking the
formulation during 10 s to 1 min; [0454] pushing the formulation
through a needle (25 G, preferably 26 G, more preferably 27 G, even
more preferably 28 G, most preferably 29 G or more) with a syringe
(the syringe used can be any commercially available syringe, such
as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL, 30 mL, 40 mL
up to 50 mL syringe); [0455] rotating for two days at 10 rpm;
and/or [0456] stirring for 1 hour at room temperature and/or 4-48
hours at 4.degree. C. at at least 10 rpm (such as 50 rpm, 100 rpm
or more).
[0457] An example of a preferred formulation of the invention with
these characteristics comprises 10 mg/mL of the polypeptide of the
invention, 15 mM histidine pH 6.5, 8% sucrose and 0.01% Tween 80.
Another example of a preferred formulation of the invention with
these characteristics comprises 10 mg/mL of the polypeptide of the
invention, 10 mM histidine pH 6.0, 10% sucrose and 0.005% Tween
80.
[0458] General methods for producing the single variable domains
and/or polypeptides present in the formulation of the invention are
known to the skilled person and/or have been described in the art.
The single variable domains and/or polypeptides can be produced in
any host known to the skilled person. For example but without being
limiting, the single variable domains and/or polypeptides can be
produced in prokaryotic hosts among which E. coli or eukaryotic
hosts, for example eukaryotic host selected from insect cells,
mammalian cells, and lower eukaryotic hosts comprising yeasts such
as Pichia, Hansenula, Saccharomyces, Kluyveromyces, Candida,
Torulopsis, Torulaspora, Schizosaccharomyces, Citeromyces,
Pachysolen, Debaromyces, Metschunikowia, Rhodosporidium,
Leucosporidium, Botryoascus, Sporidiobolus, Endomycopsis,
preferably Pichia pastoris. Production of Nanobodies in prokaryotes
and lower eukaryotic hosts such as Pichia pastoris has e.g. been
described in WO 94/04678, WO 94/25591 and WO 08/142,164. The
contents of these applications are explicitly referred to in the
connection with general culturing techniques and methods, including
suitable media and conditions. The contents of these documents are
incorporated by reference. The skilled person can also devise
suitable genetic constructs for expression of the polypeptides of
the invention in different hosts on the basis of the present
application and common general knowledge. The present invention
also relates to conditions and genetic constructs described in the
art, for example the general culturing methods, plasmids, promoters
and leader sequences described in WO 94/25591, WO 08/020,079.
Gasser et al. 2006 (Biotechnol. Bioeng. 94: 535); Gasser et al.
2007 (Appl. Environ. Microbiol. 73: 6499); or Damasceno et al. 2007
(Microbiol. Biotechnol. 74: 381).
[0459] More particularly, the method for the expression and/or
production of a polypeptide comprising one or more single variable
domains at least comprising the steps of: [0460] a) cultivating a
host or host cell (as defined herein) under conditions that are
such that said host or host cell will multiply; [0461] b)
maintaining said host or host cell under conditions that are such
that said host or host cell expresses and/or produces the
polypeptide; [0462] c) isolating and/or purifying the secreted
polypeptide from the medium.
[0463] To produce/obtain expression of the polypeptide, the
transformed host cell or transformed host organism may generally be
kept, maintained and/or cultured under conditions such that the
(desired) polypeptide is expressed/produced. Suitable conditions
will be clear to the skilled person and will usually depend upon
the host cell/host organism used, as well as on the regulatory
elements that control the expression of the (relevant) nucleotide
sequence. Again, reference is made to the handbooks and patent
applications mentioned above.
[0464] Generally, suitable conditions may include the use of a
suitable medium, the presence of a suitable source of food and/or
suitable nutrients, the use of a suitable temperature, and
optionally the presence of a suitable inducing factor or compound
(e.g. when the nucleotide sequences of the invention are under the
control of an inducible promoter); all of which may be selected by
the skilled person. Again, under such conditions, the amino acid
sequences of the invention may be expressed in a constitutive
manner, in a transient manner, or only when suitably induced.
[0465] The polypeptide of the invention may then be isolated from
the host cell/host organism and/or from the medium in which said
host cell or host organism was cultivated, using protein isolation
and/or purification techniques known per se, such as (preparative)
chromatography and/or electrophoresis techniques, differential
precipitation techniques, affinity techniques (e.g. using a
specific, cleavable amino acid sequence fused with the polypeptide
of the invention) and/or preparative immunological techniques (i.e.
using antibodies against the polypeptide to be isolated).
[0466] In the present invention, the host can be removed from the
culture medium by routine means. For example, the host can be
removed by centrifugation or filtration. The solution obtained by
removal of the host from the culture medium is also referred to as
culture supernatant, or clarified culture supernatant. The
polypeptides of the invention can be purified from the culture
supernatant by standard methods. Standard methods include, but are
not Limited to chromatographic methods, including size exclusion
chromatography, hydrophobic interaction chromatography, ion
exchange chromatography, and affinity chromatography. These methods
can be performed alone or in combination with other purification
methods, e.g. precipitation or gel electrophoresis. The skilled
person can devise suitable combinations of purification methods for
the polypeptides of the invention on the basis of common general
knowledge. For specific examples the art cited herein is referred
to.
[0467] in one exemplary embodiment, the polypeptides of the
invention can be purified from culture supernatant by a combination
of affinity chromatography on Protein A, ion exchange
chromatography and size exclusion chromatography. Reference to any
"step of purification", includes, but is not limited to these
particular methods.
[0468] More specifically, the polypeptides of the invention can be
purified from culture supernatant using a process wherein the
clarified supernatant (obtained by centrifugation) is captured on
any combination of columns selected from (without being limiting)
affinity chromatography resin such as Protein A resin, Cation
Exchange Chromatography (CIEC) or an Anion Exchange Chromatography
(ALEC) using for example Poros 50HS(POROS), SOURCE 30S or SOURCE
15S (GE Healthcare), SP Sepharose (GE Healthcare), Capto S (GE
Healthcare), Capto MMC (GE Healthcare) or Poros 50HQ (POROS),
SOURCE 30Q or SOURCE 15Q (GE Healthcare), Q Sepharose (GE
Healthcare), Capto Q and DEAE Sepharose (GE Healthcare), Size
exclusion chromatography (SE-HPLC) using for example Superdex 75 or
Superdex 200 (GE Healthcare), hydrophobic interaction
chromatography (HIC) using for example octyl, butyl sepharose or
equivalents, optionally also including a tangential flow filtration
(TFF) step. Any combination of columns can be used for the
purification of the polypeptides of the invention, such as e.g.
Protein A resin followed by Cation Exchange Chromatography or two
Cation Exchange Chromatography steps.
[0469] The present invention also provides methods for preparing
the stable formulations of the invention comprising the
polypeptides of the invention. More particularly, the present
invention provides methods for preparing stable formulations of
such polypeptides, said methods comprising concentrating a fraction
containing the purified polypeptide to the final polypeptide
concentration using e.g. a semipermeable membrane with an
appropriate molecular weight (MW) cutoff (e.g. a 5 kD cutoff for
single variable domains; a 10 kD cutoff for bivalent polypeptides
comprising two single variable domains; or a 15 kD cutoff for
trivalent polypeptides comprising three single variable domains)
and diafiltering and/or ultrafiltering to buffer exchange and
further concentrate the polypeptide fraction into the formulation
buffer using the same membrane. As extensively described above, the
formulation buffer of the present invention may further comprise at
least one of: [0470] a) A buffer at a concentration of 10 mM to 100
mM selected from the group consisting of hepes pH 7.0-8.0,
histidine pH 6.0-6.5, MES pH 6.0 and acetate pH 5.5-6.0; [0471] b)
An excipient at a concentration of 1% to 20%; [0472] c) A
surfactant at a concentration of 0.001% to 1% selected from Tween
80, Tween 20 or a poloxamer.
[0473] The pH of the formulation may range from about 5.5 to about
8.0, or may range from about 6.0 to about 7.5, preferably from
about 6.2 to 7.5, from about 6.2 to 7.0, most preferably from about
6.5 to 7.0.
[0474] Surfactant (e.g. Tween 20, Tween 80 or poloxamer) will be
added after the final diafiltration/ultrafiltration step at a
concentration in the range of about 0% to 1%, preferably 0.001% to
0.1%, or 0.01% to 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%,
0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably 0.01% or
0.005%.
[0475] The formulation of the present invention may be sterilized
by various sterilization methods, including sterile filtration,
radiation, etc. In a specific embodiment, the polypeptide
formulation is filter-sterilized with a presterilized 0.2 micron
filter.
[0476] Preferably, the formulation of the present invention is
supplied in a hermetically sealed container. Liquid formulations
may comprise a quantity between 1 mL and 20 mL, preferably about 1
mL, 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL,
or 20 mL.
[0477] The formulation of the present invention can be prepared as
unit dosage forms by preparing a vial containing an aliquot of the
formulation for a one time use. For example, a unit dosage of
liquid formulation per vial may contain 1 mL, 2 mL, 3 mL, 4 mL, 5
mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 15 mL, or 20 mL of the
formulation. The pharmaceutical unit dosage forms can be made
suitable for any form of delivery of the polypeptide of the
invention including (without being limiting) parenteral delivery,
topical delivery, pulmonary delivery, intranasal delivery, vaginal
delivery, enteral delivery, rectal delivery, oral delivery and/or
sublingual delivery. In one aspect, the present invention relates
to a pharmaceutical unit dosage form suitable for parenteral (such
as e.g. intravenous, intraarterial, intramuscular, intracerebral,
intraosseous, intradermal, intrathecal, intraperitoneal,
subcutaneous, etc.) administration to a subject, comprising a
formulation of the invention in a suitable container. In another
preferred aspect, the subject is a human. In a specific embodiment,
the formulations of the present invention are formulated into
single dose vials as a sterile liquid that contains 10 mg/mL of one
of SEQ ID NO's: 1 to 6, 15 mM histidine buffer at pH 6.5, 8%
sucrose and 0.01% Tween 80. In another specific embodiment, the
formulations of the present invention are formulated into single
dose vials as a sterile liquid that contains 10 mg/mL of one of SEQ
ID NO's: 1 to 6, 10 mM histidine buffer at pH 6.0, 10% sucrose and
0.005% Tween 80.
[0478] The amount of a formulation of the present invention which
will be effective in the prevention, treatment and/or management of
a certain disease or disorder can be determined by standard
clinical techniques well-known in the art or described herein. The
precise dose to be employed in the formulation will also depend on
the route of administration, and should be decided according to the
judgment of the practitioner and each patient's circumstances.
Effective doses may be extrapolated from dose-response curves
derived from in vitro or animal model test systems. For
formulations of the polypeptide, encompassed by the invention, the
dosage administered to a patient may further be calculated using
the patient's weight in kilograms (kg) multiplied by the dose to be
administered in mg/kg.
[0479] The required volume (in mL) to be given is then determined
by taking the mg dose required divided by the concentration of the
polypeptide formulation. The final calculated required volume will
be obtained by pooling the contents of as many vials as are
necessary into syringe(s) to administer the polypeptide formulation
of the invention.
[0480] The present invention also encompasses a finished packaged
and labelled pharmaceutical product. This article of manufacture or
kit includes the appropriate unit dosage form in an appropriate
vessel or container such as a glass vial or other container that is
hermetically sealed. In one embodiment, the unit dosage form is
suitable for intravenous, intramuscular, intranasal, oral, topical
or subcutaneous delivery. Thus, the invention encompasses
formulations, preferably sterile, suitable for each delivery route.
In the case of dosage forms suitable for parenteral administration
(such as e.g. subcutaneous administration) the active ingredient,
e.g., polypeptide of the invention, is sterile and suitable for
administration as a particulate free solution.
[0481] As with any pharmaceutical product, the packaging material
and container are designed to protect the stability of the product
during storage and shipment. Further, the products of the invention
include instructions for use or other informational material that
advise the physician, technician or patient on how to appropriately
prevent or treat the disease or disorder in question. In other
words, the article of manufacture includes instruction means
indicating or suggesting a dosing regimen including, but not
limited to, actual doses, monitoring procedures, and other
monitoring information.
[0482] Specifically, the invention provides an article of
manufacture comprising packaging material, such as a box, bottle,
tube, vial, container, sprayer, insufflator, intravenous (i.v.)
bag, envelope and the like; and at least one unit dosage form of a
pharmaceutical agent contained within said packaging material,
wherein said pharmaceutical agent comprises the formulation
containing the polypeptide. The packaging material includes
instruction means which indicate that said polypeptide can be used
to prevent, treat and/or manage one or more symptoms associated
with the disease or disorder by administering specific doses and
using specific dosing regimens as described herein.
[0483] The invention also provides an article of manufacture
comprising packaging material, such as a box, bottle, tube, vial,
container, sprayer, insufflator, intravenous (i.v.) bag, envelope
and the like; and at least one unit dosage form of each
pharmaceutical agent contained within said packaging material,
wherein one pharmaceutical agent comprises a formulation containing
the polypeptide of interest, and wherein said packaging material
includes instruction means which indicate that said agents can be
used to prevent, treat and/or manage the disease or disorder by
administering specific doses and using specific dosing regimens as
described herein.
[0484] The invention also provides an article of manufacture
comprising packaging material, such as a box, bottle, tube, vial,
container, sprayer, insufflator, intravenous (i.v.) bag, envelope
and the like; and at least one unit dosage form of each
pharmaceutical agent contained within said packaging material,
wherein one pharmaceutical agent comprises a formulation containing
the polypeptide, and wherein said packaging material includes
instruction means which indicate that said agents can be used to
prevent, treat and/or manage one or more symptoms associated with
the disease or disorder by administering specific doses and using
specific dosing regimens as described herein.
[0485] The formulations, containers, pharmaceutical unit dosages
and kits of the present invention may be administered to a subject
to prevent, treat and/or manage a specific disease and/or disorder.
In a specific aspect, the formulations, containers, pharmaceutical
unit dosages and kits of the present invention are administered to
a subject to prevent, treat and/or manage a disease and/or disorder
associated with or characterized by aberrant expression and/or
activity of a certain target or one or more symptoms thereof. In
another specific aspect, the formulations, containers,
pharmaceutical unit dosages and kits of the present invention are
administered to a subject to prevent, treat and/or manage diseases
and/or disorders associated with aberrant expression and/or
activity of RANKL, diseases and/or disorders associated with
overexpression of IL-6, or diseases and disorders associated with
heterodimeric cytokines and their receptors or one or more symptoms
thereof.
[0486] Diseases and disorders associated with aberrant expression
and/or activity of RANKL are for example bone diseases and
disorders, and include (without being limiting) the following
diseases and disorders: Osteoporosis (McClung 2006, Current
Osteoporosis Reports 4: 28-33), including, but not limited to,
primary osteoporosis, endocrine osteoporosis (including, but not
limited to, hyperthyroidism, hyperparathyroidism (Anandarajah and
Schwarz 2006, J. Cell Biochem. 97: 226-232), Cushing's syndrome,
and acromegaly), hereditary and congenital forms of osteoporosis
(including, but not limited to, osteogenesis imperfecta,
homocystinuria, Menkes' syndrome, Riley-Day syndrome), osteoporosis
due to immobilization of extremities, glucocorticoid-induced
osteoporosis (Locklin et al. 2001, Bone 28 (Suppl.): S80; McClung
2006, Current Osteoporosis Reports 4: 28-33; Anandarajah and
Schwarz 2006, J. Cell Biochem. 97: 226-232) and post-menopausal
osteoporosis (McClung 2006, Current Osteoporosis Reports 4: 28-33);
(Juvenile or Familial) Paget's disease (Cundy et al. 2002, Hum.
Mol. Genet. 11: 2119-2127; Whyte et al. 2002, J. Bone Miner. Res.
17: 26-29; Whyte et al. 2002, N. Engl. J. Med. 347: 175-184;
Johnson-Pais et al. 2003, J. Bone Miner Res. 18: 376-380;
Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232;
Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232);
Osteomyelitis, i.e., an infectious lesion in bone, leading to bone
loss; Hypercalcemia (Anandarajah and Schwarz 2006, J. Cell Biochem.
97: 226-232), including, but not limited to, hypercalcemia
resulting from solid tumors (including, but not limited to, breast,
lung and kidney) and hematologic malignancies (including, but not
limited to, multiple myeloma (Sordillo and Pearse 2003, Cancer 97
(3 Suppl): 802-812; Vanderkerken et al. 2003, Cancer Res. 63:
287-289), lymphoma and leukemia), idiopathic hypercalcemia, and
hypercalcemia associated with hyperthyroidism and renal function
disorders; Bone loss, including but not limited to, osteopenia
following surgery, osteopenia induced by steroid administration,
osteopenia associated with disorders of the small and large
intestine, and osteopenia associated with chronic hepatic and renal
diseases; Osteonecrosis, i.e., bone cell death, including, but not
limited to, osteonecrosis associated with traumatic injury,
osteonecrosis associated with Gaucher's disease, osteonecrosis
associated with sickle cell anemia, osteonecrosis associated with
systemic lupus erythematosus, osteonecrosis associated with
rheumatoid arthritis, osteonecrosis associated with periodontal
disease, osteonecrosis associated with osteolytic metastasis, and
osteonecrosis associated with other condition; Bone loss associated
with arthritic disorders such as psoriatic arthritis, rheumatoid
arthritis, loss of cartilage and joint erosion associated with
rheumatoid arthritis (Bezerra et al. 2005, Brazilian Journal of
Medical and Biological Research 38: 161-170; Anandarajah and
Schwarz 2006, J. Cell Biochem. 97: 226-232); Arthritis (Bezerra et
al. 2005, Brazilian Journal of Medical and Biological Research 38:
161-170), including inflammatory arthritis (McClung 2006, Current
Osteoporosis Reports 4: 28-33), Collagen-induced arthritis (Bezerra
et al. 2005, Brazilian Journal of Medical and Biological Research
38: 161-170); Periprosthetic osteolysis (McClung 2006, Current
Osteoporosis Reports 4: 28-33; Anandarajah and Schwarz 2006, J.
Cell Biochem. 97: 226-232); Cancer-related bone disease (McClung
2006, Current Osteoporosis Reports 4: 28-33); Bone loss associated
with aromatase inhibitor therapy (Lewiecki 2006, Expert Opin. Biol.
Ther. 6: 1041-1050); Bone loss associated with androgen deprivation
therapy (Lewiecki 2006, Expert Opin. Biol. Ther. 6: 1041-1050);
Bone loss associated bone metastasis; Bone loss associated with
diseases having immune system involvement, such as adult and
childhood leukaemias, cancer metastasis, autoimmunity, and various
viral infections (Holstead Jones et al. 2002, Ann. Rheum. Dis. 61
(Suppl II): ii32-ii39); Osteopenic disorders such as adult and
childhood leukaemia (Oliveri et al. 1999, Henry Ford Hosp. Med. 39:
45-48); chronic infections such as hepatitis C or HIV (Stellon et
al. 1985, Gastroenterology 89: 1078-1083); autoimmune disorders
such as diabetes mellitus (Piepkorn et al. 1997, Horm. Metab. Res.
29: 584-91), and lupus erythematosus (Seitz et al. 1985, Ann. Rheum
Dis, 44: 4.38-445); allergic diseases such as asthma (Ebeling et
al. 1998, J. Bone Min. Res. 13: 1283-1289); lytic bone metastases
in multiple cancers such as breast cancer (Coleman 1998, Curr.
Opin. Oncol. 10 (Suppl 1): 7-13); Prostate cancer; Myeloma bone
disease (Anandarajah and Schwarz 2006, J. Cell Biochem. 97:
226-232); Periodontal infections (Anandarajah and Schwarz 2006, J.
Cell Biochem. 97: 226-232); Expansile skeletal hyperphosphatasia
(Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232); Bone
metastases (Lewiecki 2006, Expert Opin. Biol. Ther. 6: 1041-1050;
Anandarajah and Schwarz 2006, J. Cell Biochem. 97: 226-232).
[0487] Also encompassed within the scope of the present invention
is the prevention and/or treatment with the formulations,
containers, pharmaceutical unit dosages and kits of the invention
of other diseases and disorders associated with an imbalance in the
RANKL/RANK/OPG pathway. Such diseases and disorders include but are
not limited to osteoporosis, inflammatory conditions, autoimmune
conditions, asthma, rheumatoid arthritis, multiple sclerosis,
Multiple myeloma (Sordillo and Pearse 2003, Cancer 97 (3 Suppl):
802-812; Vanderkerken et al. 2003, Cancer Res. 63: 287-289);
Vascular diseases (Anandarajah and Schwarz 2006, J. Cell Biochem.
97: 226-232) and Cardiovascular disease (Lewiecki 2006, Expert
Opin. Biol. Ther. 6: 1041-1050).
[0488] Also encompassed within the scope of the present invention
is the prevention and/or treatment with the formulations,
containers, pharmaceutical unit dosages and kits of the invention
of diseases and disorders associated with osteopetrosis such as
osteopetrosis tarda, osteopetrosis congenita and marble bone
disease.
[0489] Disease and disorders caused by aberrant expression and or
activity, such as excessive IL-6 production or signaling include
sepsis (Starnes et al., 1999) and various forms of cancer such as
multiple myeloma disease (MM), renal cell carcinoma (RCC), plasma
cell leukaemia (Klein et al., 1991), lymphoma,
B-lymphoproliferative disorder (BLPD) and prostate cancer.
Non-limiting examples of other diseases caused by aberrant
expression and/or activity, such as excessive IL-6 production or
signalling include bone resorption (osteoporosis) (Roodman at al.,
1992; Jilka at al., 1992), cachexia (Strassman et al., 1992),
psoriasis, mesangial proliferative glomerulonephritis, Kaposi's
sarcoma, AIDS-related lymphoma (Emilie et al., 1994), inflammatory
diseases and disorder such as rheumatoid arthritis, systemic onset
juvenile idiopathic arthritis, hypergammaglobulinemia (Grau et al.,
1990), Crohn's disease, ulcerative colitis, systemic lupus
erythematosus (SLE), multiple sclerosis, Castleman's disease, IgM
gammopathy, cardiac myxoma, asthma (in particular allergic asthma)
and autoimmune insulin-dependent diabetes mellitus (Campbell et
al., 1991).
[0490] Diseases and disorders associated with heterodimeric
cytokines and their receptors include inflammation and inflammatory
disorders such as bowel diseases (colitis, Crohn's disease, IBD),
infectious diseases, psoriasis, cancer, autoimmune diseases (such
as MS), carcoidis, transplant rejection, cystic fibrosis, asthma,
chronic obstructive pulmonary disease, rheumatoid arthritis, viral
infection, common variable immunodeficiency.
[0491] The formulations, containers, pharmaceutical unit dosages
and kits of the present invention may also be advantageously
utilized in combination with one or more other therapies (e.g., one
or more other prophylactic or therapeutic agents), preferably
therapies useful in the prevention, treatment and/or management of
the (same or another) disease or disorder. When one or more other
therapies (e.g., prophylactic or therapeutic agents) are used, they
can be administered separately, in any appropriate form and by any
suitable route. Therapeutic or prophylactic agents include, but are
not limited to, small molecules, synthetic drugs, peptides,
polypeptides, proteins, nucleic acids (e.g., DNA and RNA
nucleotides including, but not limited to, antisense nucleotide
sequences, triple helices, RNAi, and nucleotide sequences encoding
biologically active proteins, polypeptides or peptides),
antibodies, other single variable domains, synthetic or natural
inorganic molecules, mimetic agents, and synthetic or natural
organic molecules. Any therapy (e.g., prophylactic or therapeutic
agents) which is known to be useful, or which has been used or is
currently being used for the prevention, treatment and/or
management of one or more symptoms associated with a specific
disease or disorder, can be used in combination with the
formulations of the present invention in accordance with the
invention described herein.
[0492] A formulation of the invention may be administered to a
mammal, preferably a human, concurrently with one or more other
therapies (e.g., one or more other prophylactic or therapeutic
agents). The term "concurrently" is not limited to the
administration of prophylactic or therapeutic agents/therapies at
exactly the same time, but rather it is meant that the formulation
of the invention and the other agent/therapy are administered to a
mammal in a sequence and within a time interval such that the
polypeptide contained in the formulation can act together with the
other agent/therapy to provide an increased benefit than if they
were administered otherwise. For example, the formulation of the
invention and the one or more other prophylactic or therapeutic
agents may be administered at the same time or sequentially in any
order at different points in time; however, if not administered at
the same time, they should be administered sufficiently close in
time so as to provide the desired therapeutic or prophylactic
effect.
[0493] When used in combination with other therapies (e.g.,
prophylactic and/or therapeutic agents), the formulations of the
invention and the other therapy can act additively or
synergistically. The invention contemplates administration of a
formulation of the invention in combination with other therapies
(e.g., prophylactic or therapeutic agents) by the same or different
routes of administration, e.g., oral and parenteral.
[0494] Various delivery systems are known and can be used to
administer the formulation of the present invention. Methods of
administering formulations of the present invention include, but
are not limited to, parenteral administration (e.g., intradermal,
intramuscular, intraperitoneal, intravenous and, preferably
subcutaneous), epidural administration, topical administration, and
mucosal administration (e.g., intranasal and oral routes). In a
specific embodiment, liquid formulations of the present invention
are administered parenteral.
[0495] The invention will now be further described by means of the
following non-limiting preferred aspects and examples:
Preferred Aspects
[0496] 1. A formulation, such as a pharmaceutical formulation,
comprising an aqueous carrier having a pH of 5.5 to 8.0 and a
polypeptide comprising one or more single variable domains at a
concentration of 1 mg/mL to 200 mg/mL, said formulation being
formulated for administration to a human subject and said
formulation further comprising one or more components selected
from: [0497] a) A buffer at a concentration of 10 mM to 100 mM
selected from the group consisting of histidine pH 6.0-6.5, hepes
pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0; [0498] b) An excipient at a concentration of 1% to 20%;
[0499] c) A surfactant at a concentration of 0.001% to 1% selected
from Tween 80, Tween 20 or a poloxamer,
[0500] wherein said formulation has an inorganic salt concentration
of 150 mM or lower, [0501] 2. The formulation of aspect 1, wherein
the inorganic salt concentration is from 50 mM to 100 mM or lower.
[0502] 3. The formulation of aspect 2, that does not contain any
inorganic salt. [0503] 4. The formulation of any of aspects 1 to 3,
wherein the formulation comprises a buffer at a concentration of 10
mM to 100 mM selected from the group consisting of histidine pH
6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and
acetate pH 5.5-6.0. [0504] 5. The formulation of aspect 4, wherein
the formulation comprises a histidine buffer pH 6.5. [0505] 6. The
formulation of aspect 4, wherein the formulation comprises a
histidine buffer pH 6.0. [0506] 7. The formulation of any of
aspects 4 to 6, wherein the buffer has a concentration of 10 to 50
mM, preferably 10 to 20 mM, such as 10 mM or 15 mM. [0507] 8. The
formulation of any of aspects 1 to 7, wherein the formulation
comprises a surfactant at a concentration of 0.001% to 1% selected
from Tween 80, Tween 20 or a poloxamer. [0508] 9. The formulation
of aspect 8, wherein the formulation comprises Tween 80. [0509] 10.
The formulation of any of aspects 8 or 9, wherein the surfactant
has a concentration of 0.001% to about 0.1%, or about 0.01% to
about 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%, 0.05%, 0.08%,
0.1%, 0.5%, or 1% of the formulation, preferably 0.01%. [0510] 11.
The formulation of any of aspects 1 to 3, wherein the formulation
comprises an excipient at a concentration of 1% to 20%. [0511] 12.
The formulation of aspect 11, wherein the excipient is a
dissaccharide and/or a polyol. [0512] 13. The formulation of aspect
11, wherein the excipient is selected from sucrose, mannitol,
sorbitol and trehalose. [0513] 14. The formulation of any of
aspects 11 to 13, wherein the excipient has a concentration of 2.5%
to 15%, preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%. [0514]
15. The formulation of any of aspects 1 to 14, wherein the
polypeptide has a solubility of at least 20 mg/mL as determined by
the PEG exclusion method or by centrifugal ultrafiltration. [0515]
16. The formulation of aspect 15, wherein the polypeptide has a
solubility of at least 50 mg/mL as determined by the PEG exclusion
method or by centrifugal ultrafiltration. [0516] 17. The
formulation of aspect 16, wherein the polypeptide has a solubility
of at least 90 mg/mL as determined by the PEG exclusion method or
by centrifugal ultrafiltration. [0517] 18. The formulation of
aspect 17, wherein the polypeptide has a solubility of at least 120
mg/mL as determined by the PEG exclusion method or by centrifugal
ultrafiltration. [0518] 19. The formulation of aspect 18, wherein
the polypeptide has a solubility of at least 150 mg/mL as
determined by the PEG exclusion method or by centrifugal
ultrafiltration. [0519] 20. The formulation of aspect 19, wherein
the polypeptide has a solubility of at least 200 mg/mL as
determined by the PEG exclusion method or by a concentration
experiment. [0520] 21. The formulation of any of aspects 1 to 20,
wherein the formulation at least comprises one or more components
selected from: [0521] a) A buffer at a concentration of 10 mM to
100 mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0; [0522] c) A surfactant at a concentration of 0.001% to 1%
selected from Tween 80, Tween 20 or a poloxamer. [0523] 22. The
formulation of aspect 21, wherein the formulation comprises a
buffer at a concentration of 10 mM to 100 mM selected from the
group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH
6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0. [0524] 23. The
formulation of aspect 22, wherein the formulation comprises a
histidine buffer pH 6.5. [0525] 24. The formulation of aspect 22,
wherein the formulation comprises a histidine buffer pH 6.0. [0526]
25. The formulation of any of aspects 22 to 24, wherein the buffer
has a concentration of 10 to 50 mM, preferably 10 to 20 mM, such as
10 mM or 15 mM. [0527] 26. The formulation of any of aspects 21 to
25, wherein the formulation comprises a surfactant at a
concentration of 0.001% to 1% selected from Tween 80, Tween 20 or a
poloxamer. [0528] 27. The formulation of aspect 26, wherein the
formulation comprises Tween 80, [0529] 28. The formulation of any
of aspects 26 or 27, wherein the surfactant has a concentration of
0.001% to about 0.1%, or about 0.01% to about 0.1% such as 0.001%,
0.005%, 0.01%, 0.02%, 0.05%, 0.08%, 0.1%, 0.5%, or 1% of the
formulation, preferably 0.01%. [0530] 29. The formulation of any of
aspect 1 to 28, wherein the polypeptide has a melting temperature
of at least 59.degree. C. or more (such as 59.5.degree. C. or
more), preferably at least 60.degree. C. or more (such as
60.5.degree. C. or more), more preferably at least 61.degree. C. or
more (such as 61.5.degree. C. or more) or at least 62.degree. C. or
more (such as 62.5.degree. C. or more), most preferably at least
63.degree. C. or more (such as 63.5.degree. C. or more) as measured
by the thermal shift assay (TSA) and/or differential scanning
calorimetry (DSC). [0531] 30. The formulation of aspect 29, which
has a pH of 6.2 to 7.5. [0532] 31. The formulation of aspect 30,
which has a pH of 6.5 to 7.5. [0533] 32. The formulation of aspect
31, which has a pH of 6.5 to 7.0. [0534] 33. The formulation of any
of aspects 29 to 32, wherein the formulation at least comprises one
or more components. [0535] a) A buffer at a concentration of 10 mM
to 100 mM selected from the group consisting of histidine pH
6.0-6.5, hepes pH 7.0-8.0, MES pH6.0, succinate pH 6.0-6.5 and
acetate pH 5.5-6.0; [0536] b) An excipient at a concentration of 1%
to 20%. [0537] 34. The formulation of aspect 33, wherein the
formulation comprises a buffer at a concentration of 10 mM to 100
mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0. [0538] 35. The formulation of aspect 34, wherein the
formulation comprises a histidine buffer pH 6.0-6.5 or a hepes
buffer pH 7.0. [0539] 36. The formulation of any of aspects 34 or
35, wherein the buffer has a concentration of 10 to 50 mM,
preferably 10 to 20 mM, such as 10 mM or 15 mM. [0540] 37. The
formulation of any of aspects 33 to 36, wherein the formulation
comprises an excipient at a concentration of 1% to 20%. [0541] 38.
The formulation of aspect 37, wherein the excipient is a
dissaccharide and/or a polyol. [0542] 39. The formulation of aspect
38, wherein the excipient is selected from sucrose, mannitol,
sorbitol and trehalose. [0543] 40. The formulation of any of
aspects 37 to 39, wherein the excipient has a concentration of 2.5%
to 15%, preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%. [0544]
41. The formulation of any of aspects 1 to 40, wherein the
polypeptide is stable after multiple (up to 10) freeze/thaw cycles,
said stability as determined by SE-HPLC, IEX-HPLC, RP-HPLC, Biacore
analysis and/or potency assay. [0545] 42. The formulation of any of
aspects 1 to 41, wherein the polypeptide is stable during storage
at a temperature of 2-8.degree. C. up to up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more), said stability as
determined by OD320/OD280 measurement, elastic light scattering,
SE-HPLC and/or RP-HPLC. [0546] 43. The formulation of aspects 42,
wherein no particulates are present as measured by OD320/OD280
measurement and/or elastic light scattering. [0547] 44. The
formulation of any of aspects 42 or 43, in which the OD320/OD280 is
0.05 or less. [0548] 45. The formulation of any of aspects 42 or
43, in which the scattering in elastic light scattering stays
within the detection range, and/or preferably is 1000 abs or less.
[0549] 46. The formulation of any of aspects 42 to 45, wherein the
formulation comprises a buffer at a concentration of 10 mM to 100
mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0. [0550] 47. The formulation of aspect 46, wherein the
formulation comprises a histidine buffer pH 6.0-6.5 or a hepes
buffer pH 7.0. [0551] 48. The formulation of any of aspects 46 or
47, wherein the buffer has a concentration of 10 to 50 mM,
preferably 10 to 20 mM, such as 10 mM or 15 mM. [0552] 49. The
formulation of any of aspects 1 to 48, wherein the polypeptide is
stable during storage at a temperature of 37.+-.5.degree. C. up to
at least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more), said
stability as determined by OD320/OD280 measurement, elastic light
scattering, SE-HPLC, RP-HPLC, IEX-HPLC, potency assay (such as
Biacore or ELISA) and/or SOS-PAGE. [0553] 50. The formulation of
aspect 49, wherein less than 10% (preferably less than 8%, more
preferably less than 7%, most preferably less than 5%) of the
polypeptides forms pyroglutamate at the N-terminal glutamic acid
during storage, the % of pyroglutamate as measured by RP-HPLC.
[0554] 51. The formulation of aspect 50, which has a pH of 7.0 or
less. [0555] 52. The formulation of any of aspects 50 or 51,
wherein the formulation comprises a buffer at a concentration of 10
mM to 100 mM selected from the group consisting of histidine pH
6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and
acetate pH 5.5-6.0. [0556] 53. The formulation of aspect 52,
wherein the formulation comprises a histidine buffer or an acetate
buffer. [0557] 54. The formulation of any of aspects 52 or 53,
wherein the buffer has a concentration of 10 to 50 mM, preferably
20 to 20 mM, such as 10 mM or 15 mM. [0558] 55. The formulation of
any of aspects 49 to 54, wherein less than 10% (preferably less
than 7.5%, more preferably less than 5%, most preferably less than
2%) of the polypeptides forms dimers during storage, the % of
dimers as measured by SE-HPLC. [0559] 56. The formulation of aspect
55, wherein the formulation at least comprises one or more
components selected from: [0560] a) A buffer at a concentration of
10 mM to 100 mM selected from the group consisting of histidine pH
6.0-6.5, hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and
acetate pH 5.5-6.0; [0561] b) An excipient at a concentration of 1%
to 20%. [0562] 57. The formulation of aspect 56, wherein the
formulation comprises a buffer at a concentration of 10 mM to 100
mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0. [0563] 58. The formulation of aspect 57, wherein the
formulation comprises a histidine buffer or an acetate buffer.
[0564] 59. The formulation of any of aspects 57 or 58, wherein the
buffer has a concentration of 10 to 50 mM, preferably 10 to 20 mM,
such as 10 mM or 15 mM. [0565] 60. The formulation of any of
aspects 55 to 59, wherein the formulation comprises an excipient at
a concentration of 1% to 20%. [0566] 61. The formulation of aspect
60, wherein the excipient is a disaccharide and/or a polyol. [0567]
62. The formulation of aspect 60, wherein the excipient is a
non-reducing sugar. [0568] 63. The formulation of aspect 61 or 62,
wherein the excipient is selected from trehalose, mannitol and
sucrose. [0569] 64. The formulation of any of aspects 60 to 63,
wherein the excipient has a concentration of 2.5% to 15%,
preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%. [0570] 65. The
formulation of any of aspects 49 to 64, wherein no particulates are
present as measured by OD320/OD280 measurement and/or elastic light
scattering. [0571] 66. The formulation of aspect 65, in which the
OD320/OD280 is 0.05 or less. [0572] 67. The formulation of aspect
65, in which the scattering in elastic light scattering stays
within the detection range, and/or preferably is 1000 abs or less.
[0573] 68. The formulation of any of aspects 65 to 67, wherein the
formulation comprises a buffer at a concentration of 10 mM to 100
mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0. [0574] 69. The formulation of aspect 68, wherein the
formulation comprises a histidine buffer. [0575] 70. The
formulation of any of aspects 68 or 69, wherein the buffer has a
concentration of 10 to 50 mM, preferably 10 to 20 mM, such as 10 mM
or 15 mM. [0576] 71. The formulation of any of aspects 49 to 70,
wherein at least 80% (preferably at least 90%, more preferably at
least 95% or even at least 99%) of the polypeptides retain their
binding activity to at least one of their targets after storage
compared to the binding activity prior to storage, said binding
activity as measured by ELISA and/or Biacore. [0577] 72. The
formulation of aspect 71, wherein the formulation at least
comprises one or more components selected from: [0578] a) A buffer
at a concentration of 10 mM to 100 mM selected from the group
consisting of hepes pH 7.0-8.0, histidine pH 6.0-6.5, MES pH 6.0
and acetate pH 5.5-6.0; [0579] b) An excipient at a concentration
of 1% to 20%. [0580] 73. The formulation of aspect 72, wherein the
formulation comprises a buffer at a concentration of 10 mM to 100
mM selected from the group consisting of histidine pH 6.0-6.5,
hepes pH 7.0-8.0, MES pH 6.0, succinate pH 6.0-6.5 and acetate pH
5.5-6.0. [0581] 74. The formulation of aspect 73, wherein the
formulation comprises a histidine buffer. [0582] 75. The
formulation of any of aspects 73 or 74, wherein the buffer has a
concentration of 10 to 50 mM, preferably 10 to 20 mM, such as 10 mM
or 15 mM. [0583] 76. The formulation of any of aspects 71 to 75,
wherein the formulation comprises an excipient at a concentration
of 1% to 20%, [0584] 77. The formulation of aspect 76, wherein the
excipient is a disaccharide and/or a polyol. [0585] 78. The
formulation of aspect 76, wherein the excipient is a non-reducing
sugar. [0586] 79. The formulation of aspect 77 or 78, wherein the
excipient is selected from mannitol, trehalose and sucrose. [0587]
80. The formulation of any of aspects 76 to 79, wherein the
excipient has a concentration of 2.5% to 15%, preferably 5% to 10%,
such as 5%, 7.5%, 8% or 10%. [0588] 81. The formulation of any of
aspects 1 to 80, wherein the single variable domain is stable under
mechanical stress as determined by visual inspection and/or
OD320/OD280 measurement. [0589] 82. The formulation of aspect 81,
in which the OD320/OD280 is 0.05 or less. [0590] 83. The
formulation of any of aspects 81 or 82, wherein the mechanical
stress is selected from shaking during 10 s to 1 min, pushing
through a needle (25 G, preferably 26 G, more preferably 27 G, even
more preferably 28 G, most preferably 29 G or more) with a syringe,
rotation for two days at 10 rpm, and stirring for 1 hour at room
temperature and/or 4-48 hours at 4
.degree. C. at at least 10 rpm (such as 50 rpm, 100 rpm or more).
[0591] 84. The formulation of any of aspects 81 to 83, wherein the
formulation at least comprises one or more components selected
from: [0592] b) An excipient at a concentration of 1% to 20%;
[0593] c) A surfactant at a concentration of 0.001% to 1% selected
from Tween 80, Tween 20 or a poloxamer. [0594] 85. The formulation
of any of aspects 81 to 84, wherein the formulation comprises an
excipient at a concentration of 1% to 20%. [0595] 86. The
formulation of aspect 85, wherein the excipient is a sugar and/or
polyol. [0596] 87. The formulation of aspect 85, wherein the
excipient is selected from mannitol, glycine and sucrose, such as
sucrose, or mannitol and glycine. [0597] 88. The formulation of any
of aspects 85 to 87, wherein the excipient has a concentration of
2.5% to 15%, preferably 5% to 10%, such as 5%, 7.5%, 8% or 10%.
[0598] 89. The formulation of any of aspects 84 to 88, wherein the
formulation comprises a surfactant at a concentration of 0.001% to
1% selected from Tween 80, Tween 20 or a poloxamer, [0599] 90. The
formulation of aspect 89, wherein the formulation comprises Tween
80. [0600] 91. The formulation of any of aspects 89 or 90, wherein
the surfactant has a concentration of 0.001% to about 0.1%, or
about 0.01% to about 0.1% such as 0.001%, 0.005%, 0.01%, 0.02%,
0.05%, 0.08%, 0.1%, 0.5%, or 1% of the formulation, preferably
0.01%. [0601] 92. The formulation of any of aspects 1 to 91,
wherein the concentration of polypeptide is about 1 to 200 mg/ml or
more, preferably about 5 to 100 mg/mL or more, more preferably
about 5 to 50 mg/mL or more, most preferably about 5 to 30 mg/mL or
more, such as around 5 mg/mL, around 10 mg/mL, around 20 mg/mL,
around 30 mg/mL, around 40 mg/mL, around 50 mg/mL, around 60 mg/mL,
around 70 mg/mL, around 80 mg/mL, around 90 mg/mL, around 100
mg/mL, around 150 mg/mL or even more. [0602] 93. The formulation of
any of aspects 1 to 92, wherein the aqueous carrier is distilled
water. [0603] 94. The formulation of any of aspects 1 to 93,
wherein the aqueous carrier is MilliQ grade water or Water for
Injection (WFI). [0604] 95. The formulation according to any of
aspects 1 to 94, which is isotonic or slightly hypotonic. [0605]
96. The formulation according to aspect 95, which has an osmolality
of 290.+-.60 mOsm/kg. [0606] 97. The formulation of any of aspects
1 to 96, wherein the polypeptide comprises two or more single
variable domains, such as two or three. [0607] 98. The formulation
of any of aspects 1 to 97, wherein the polypeptide specifically
binds RANKL, IL-6R or IL-23. [0608] 99. The formulation of aspect
98, wherein the polypeptide is selected from one of SEQ ID NO's: 1
to 6. [0609] 100. The formulation of any of aspects 1 to 99,
wherein [0610] the polypeptide has a solubility of at least 20
mg/mL, preferably 50 mg/mL or more, more preferably 90 mg/mL or
more or 120 mg/mL or more, most preferably 150 mg/mL or more, or
even 200 mg/mL or more, as determined by the PEG exclusion method
or by a concentration experiment; [0611] the polypeptide has a
melting temperature of at least 59.degree. C. or more, preferably
at least 60.degree. C. or more, more preferably at least 61.degree.
C. or more or at least 62.degree. C. or more, most preferably at
least 63.degree. C. or more as measured by the thermal shift assay
(USA) and/or differential scanning calorimetry (DSC); [0612] no
particulates are present as measured by OD320/OD280 and/or elastic
light scattering; [0613] less than 10% of the polypeptide forms
pyroglutamate at the N-terminal glutamic acid during storage at a
temperature of 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 8 weeks,
at least 10 weeks, at least 3 months, at least 6 months, at least 1
year, 1.5 year or even 2 years or more), the % of pyroglutamate as
measured by RP-HPLC; [0614] less than 10% of the polypeptide forms
dimers during storage at a temperature of 37.+-.5.degree. C. up to
at least 2 weeks (preferably at least 3 weeks, at least 5 weeks, at
least 8 weeks, at least 10 weeks, at least 3 months, at least 6
months, at least 1 year, 1.5 year or even 2 years or more), the %
of dimers as measured by SE-HPLC; [0615] at least 80% of the
polypeptide retains its binding activity to at least one of its
targets after storage at 37.+-.5.degree. C. up to at least 2 weeks
(preferably at least 3 weeks, at least 5 weeks, at least 2 months,
at least 6 months, at least 1 year, 1.5 year or even 2 years or
more) compared to the binding activity prior to storage, said
binding activity as measured by ELISA and/or Biacore; and/or [0616]
the polypeptide is stable during mechanical stress. [0617] 101. The
formulation of any of aspects 1 to 100, wherein said formulation
comprises at least two components selected from: [0618] a) A buffer
at a concentration of 10 mM to 100 mM selected from the group
consisting of histidine pH 6.0-65, hepes pH 7.0-8.0, MES pH 6.0,
succinate pH 6.0-6.5 and acetate pH 5.5-6.0; [0619] b) An excipient
at a concentration of 1% to 20%; [0620] c) A surfactant at a
concentration of 0.001% to 1% selected from Tween 80, Tween 20 or a
poloxamer. [0621] 102. The formulation of any of aspects 1 to 101,
wherein said formulation comprises the following components: [0622]
a) A buffer at a concentration of 10 mM to 100 mM selected from the
group consisting of histidine pH 6.0-6.5, hepes pH 7.0-8.0, MES pH
6.0, succinate pH 6.0-6.5 and acetate pH 5.5-6.0; [0623] b) An
excipient at a concentration of 1% to 20%; [0624] c) A surfactant
at a concentration of 0.001% to 1% selected from Tween 80, Tween 20
or a poloxamer. [0625] 103. The formulation of any of aspects 1,
101 and/or 102, wherein the pH is between 6.0 and 8, preferably
between 6.2 and 7.5, more preferably between 6.5 and 7.5 or 6.5 and
7.0, such as 6.5 or 7.0. [0626] 104. The formulation of any of
aspects 1, 101 to 103, wherein the buffer is a histidine buffer.
[0627] 105. The formulation of aspect 104, wherein the buffer is a
histidine pH 6.5 or histidine pH 6.0 buffer. [0628] 106. The
formulation of any of aspects 104 or 105, wherein the histidine
buffer has a concentration of 10 to 50 mM, more preferably 10 to 20
mM. [0629] 107. The formulation of any of aspects 104 to 106,
wherein the histidine buffer has a concentration of about 10 mM or
about 15 mM. [0630] 108. The formulation of any of aspects 1, 101
and/or 102 to 107, wherein the excipient is a saccharide and/or
polyol. [0631] 109. The formulation of aspect 108, wherein the
excipient is a non-reducing sugar. [0632] 110. The formulation of
aspect 108, wherein the excipient is selected from mannitol,
trehalose, sorbitol and sucrose. [0633] 111. The formulation of any
of aspect 108 to 110, wherein the excipient has a concentration of
2.5% to 15%, more preferably 5% to 10%. [0634] 112. The formulation
of aspect 111, wherein the excipient has a concentration selected
from about 5%, 7.5%, 8% and 10%. [0635] 113. The formulation of any
of aspects 1, 101 and/or 102 to 112, wherein the surfactant is
Tween 80. [0636] 114. The formulation of aspect 113, wherein the
surfactant has a concentration of 0.01% to 0.1%, preferably 0.01%
to 0.05%. [0637] 115. The formulation of aspect 114, wherein the
surfactant has a concentration of about 0.01% or 0.005%. [0638]
116. The formulation of any of aspects 1 to 115, comprising: [0639]
a) A histidine pH 6.5 buffer at a concentration of 10 mM to 100 mM;
[0640] b) Sucrose at a concentration of 1% to 20%; and [0641] c)
Tween 80 at a concentration of 0.001% to 1%. [0642] 117. The
formulation of aspect 116, comprising: [0643] a) 15 mM histidine pH
6.5; [0644] b) 8% sucrose; and [0645] c) 0.01% Tween 80. [0646]
118. The formulation of aspect 117, comprising: [0647] a) 15 mM
histidine pH 6.5; [0648] b) 8% sucrose; [0649] c) 0.01% Tween 80;
and [0650] d) A polypeptide selected from SEQ ID NO's: 1 to 6.
[0651] 119. The formulation of any of aspects 1 to 115, comprising:
[0652] a) A histidine pH 6.0 buffer at a concentration of 10 mM to
100 mM; [0653] b) Sucrose at a concentration of 1% to 20%; and
[0654] c) Tween 80 at a concentration of 0.001% to 1%. [0655] 120.
The formulation of aspect 119, comprising: [0656] a) 10 mM
histidine pH 6.0; [0657] b) 10% sucrose; and [0658] c) 0.005% Tween
80. [0659] 121. The formulation of aspect 120, comprising: [0660]
a) 10 mM histidine pH 6.0; [0661] b) 10% sucrose; [0662] c) 0.005%
Tween 80; and [0663] d) A polypeptide selected from SEQ ID NO's: 1
to 6. [0664] 122. A method for the preparation of a formulation of
any of aspects 1 to 121, at least comprising the step of
concentrating the polypeptide and exchanging it with the selected
buffer and/or excipient. [0665] 123. A sealed container containing
a formulation according to any of aspects 1 to 121. [0666] 124. A
pharmaceutical unit dosage form suitable for parenteral
administration to a human, comprising a formulation according to
any of aspects 1 to 121 in a suitable container. [0667] 125. A kit
comprising one or more of the sealed containers according to aspect
123 and/or pharmaceutical unit dosage forms according to aspect
124, and instructions for use of the formulation. [0668] 126. The
formulation, container, pharmaceutical unit dosage or kit according
to any of the preceding aspects for use in therapy. [0669] 127.
Method for prevention and/or treatment of one or more diseases
and/or disorders, comprising administering to a subject in need
thereof a formulation according to any of aspects 1 to 121. [0670]
128. Method of aspect 127, wherein the disease is a disease and/or
disorder associated with aberrant expression and/or activity of
RANKL, disease and/or disorder associated with aberrant expression
and/or activity, such as overexpression of IL-6, or disease and
disorder associated with heterodimeric cytokines and their
receptors. [0671] 129. Method of aspect 128, wherein the disease is
selected from osteoporosis, cancer induced bone loss and/or bone
loss associated with autoimmunity and/or viral infection. [0672]
130. Method of aspect 128, wherein the disease is selected from
rheumatoid arthritis, abnormal synovial cell growth, plasmocytosis
induced Castleman's disease, tumor, muscle protein proteolysis,
multiple sclerosis, systemic lupus erythematosus, inflammatory
bowel disease, pancreatitis, psoriasis, angiogenesis,
systemic-onset type juvenile rheumatoid arthritis, spinal cord
injury, endothelial injury or destruction, mesothelioma,
vasculitis, osteoarthritis, inner ear disorder, cancer, rejection
after transplantation, pancreatic islet transplantation, myocardial
infraction, prostate cancer, choroidal neovascularization, muscle
regeneration, inflammatory myopathy, chronic rejection in cardiac
transplant, delayed graft function [0673] 131. Method of aspect
128, wherein the disease is selected from inflammation and
inflammatory disorders such as bowel diseases (colitis, Crohn's
disease, IBD), infectious diseases, psoriasis, cancer, autoimmune
diseases (such as MS), carcoidis, transplant rejection, cystic
fibrosis, asthma, chronic obstructive pulmonary disease, rheumatoid
arthritis, viral infection, common variable immunodeficiency.
[0674] The terms and expressions which have been employed are used
as terms of description and not of limitation, and there is no
intention in the use of such terms and expressions of excluding any
equivalents of the features shown and described or portions
thereof, it being recognized that various modifications are
possible within the scope of the invention.
[0675] All of the references described herein are incorporated by
reference, in particular for the teaching that is referenced
hereinabove.
EXAMPLES
Example 1
Formulation and Stability Studies with RANKL008a
Example 1.1
Materials and Methods Used in the Study
[0676] 1.1.1 Single variable domains
[0677] RANKL008a (SEQ ID NO: 4;
EVQLVESGGGLVQPGGSLRLSCAASGFTFSSYPMGWFRQAPGKGREFVS
SITGSGGSTYYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAAYIRPDTYLSRDYRKYDYWGQGTLV-
TVSS
GGGGSGGGSEVOLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLY-
ADSVKG
RFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGG-
GLVQPGGSLR
LSCAASGFTFSSYPMGWFRQAPGKGREFVSSITGSGGSTYYADSVKGRFTISRDNAKNTLYLQ-
MNSLRPEDTAVYYC AAYIRPDTYLSRDYRKYDYWGQGTLVTVSS) has been described
as SEQ ID NO: 759 in WO 2008/142164. RANKL008a is a trivalent
bispecific Nanobody consisting of three humanized variable domains
of a heavy-chain llama antibody, of which two identical subunits
are specific for binding to RANKL while the remaining subunit binds
to HSA. The subunits are fused head-to-tail with a G/S linker in
the following format: RANKL13H5-9GS-Alb8-9GS-RANKL13H5.
[0678] RANKL008a was expressed in Pichia pastoris and purified on
SP Sepharose as a capturing step and a Q filter as a polishing step
or on SP Sepharose as a capturing step and Capto MMC as a polishing
step or alternatively by using a ProtA capture step followed by and
SP Sepharose polishing step. Concentration of the Nanobody and
buffer switch to PBS, 10 mM phosphate+100 mM NaCl, 10 mM
phosphate+5% mannitol or 10 mM phosphate+115 mM NaCl or others
buffers was done via UF/DF or by dialysis. A final filtration on a
0.22 .mu.m filter was performed. The different batches of RANKL008a
ranged in concentration from 143 to 62.8 mg/mL. Most batches were
prepared at concentrations of about 60-85 mg/mL.
1.1.2 Other Reagents
[0679] All reagents used in the study are standard chemical
reagents of highest purity. The complete composition of D-PBS was
137 mM NaCl, 2.7 mM KCl, 10 mM Sodium Phosphate dibasic, 2 mM
Potassium Phosphate monobasic.
1.1.3 Equipment and Columns for Testing
[0680] HPLC experiments were carried out on an Agilent 1200 series
instrument from Agilent Technologies (Palo Alto, USA). The columns
used were: [0681] RP-HPLC: Zorbax 300SB-C3 5-micron; [0682]
SE-HPLC: TSKgel G2000SW.sub.XL (Tosoh Bioscience, Japan); [0683]
IEX-HPLC: Dionex ProPac .sub.wCX-10 column;
[0684] The concentration of the purified RANKL008a batches was
determined by spectroscopy at 280 nm either using a NanoDrop
ND-1000 (Thermoscientific), or a Uvikon 943 Spectrophotometer
(Kontron Instruments) or an Eppendorf Biophotometer 613.
[0685] Particle size distribution was measured on a PAMAS SVSS-C
particle counter (PArtikelMess-und AnalyseSysteme GMBH).
[0686] For potency measurements a Biacore 3000 (GE Healthcare) was
used. Elisa based potency assays were performed using standard
laboratory equipment and plate reader instrumentation.
[0687] Osmolality measurement was done with an osmometer Model 3320
from Advanced instruments.
1.1.4 Inhibition ELISA for RANKL Potency Measurement
[0688] RANKL008a interacts with human (soluble) receptor activator
of nuclear factor-kappa B ligand (RANKL) and blocks the interaction
of this ligand with its human receptor activator of nuclear
factor-kappaB (RANK), thereby preventing signalization through this
receptor. The potency of RANKL008a was measured by an ELISA-based
inhibition assay that allows assessment of the relative potency of
the RANKL binding moieties of an unknown batch of RANKL008a
relative to that of a reference batch.
[0689] For the reference, the control and the test samples,
different dilutions of Nanobodies were prepared. These dilutions
were pre-incubated with a constant amount of 5 ng/mL soluble RANKL
and a constant amount of 200 ng/mL RANK-Fc. Subsequently, this
mixture was transferred to a microtiter plate coated with a
non-blocking anti-Fc Nanobody. After washing, residual bound RANKL
was detected with a polyclonal biotinylated anti-human RANKL
antibody, followed by hors radish peroxidase (HRP)-labeled
streptavidin detection.
[0690] The relative potency of the test samples compared to the
reference samples was analysed by use of PLA 2.0 Software (Stegmann
Systems).
1.1.5 ELISA for HSA Binding
[0691] The relative potency of the HSA binding moiety in RANKL008a
was measured by an ELISA. Briefly, HSA was coated onto a plastic
multiwell Maxisorp ELISA plate by adsorption. After blocking excess
binding sites on the plates with Superbiock T20, a dilution series
of references, control and test samples was applied on the plate.
Bound Nanobody was subsequently detected using a bivalent
anti-Nanobody Nanobody, directly conjugated to horseradish
peroxidase (HRP).
[0692] The relative potency of the test samples compared to the
reference samples was analysed by use of PLA 2.0 Software (Stegmann
Systems).
1.1.6 Purity Assay of RANKL008a by Size Exclusion High Performance
Liquid Chromatography (SE-HPLC)
[0693] The SE-HPLC assay consisted of a pre-packed silica gel
TSKgel G2000SW.sub.XL column, a mobile phase consisting of KCl,
NaCl and phosphate buffer pH 7.2 (D-PBS) and UV detection at 280
nm. The relative amount of the specific protein, variant, or
impurities expressed as area %, was calculated by dividing the peak
area corresponding to the specific protein or to any protein
impurity by the total area of all integrated peaks,
1.1.7 Purity Assay of RANKL008a by Reverses Phase High Performance
Liquid Chromatography (RP-HPLC)
[0694] In the RP-HPLC assay a Zorbax 300SB-C3 column (Agilent
Technologies, Palo Alto, US) was used. The relative amount of a
specific protein impurity was determined by measuring the light
absorbance of the components eluting from the RP-HPLC column. The
relative amount of the specific protein, variant, or impurities
expressed as area %, was calculated by dividing the peak area
corresponding to the specific protein or to any protein impurity by
the total area of all integrated peaks.
1.1.8 Purity Assay of RANKL008a by Ion Exchange High Performance
Liquid Chromatography (IEX-HPLC)
[0695] The IEX-HPLC assay combined the use of a pre-packed Dionex
ProPac WCX-10 weak cation exchange column, a mobile phase
consisting of citrate buffer pH 5.5 and UV detection at 280 nm.
After loading the protein(s) on the column, bound materials were
eluted by a sodium chloride gradient. The relative amount of the
specific protein, variant, or impurities expressed as area %, was
calculated by dividing the peak area corresponding to the specific
protein or to any protein impurity by the total area of all
integrated peaks.
1.1.9 Relative Potency Determination on Biacore
[0696] RANKL or HSA was immobilized on the Biacore chip (amine
coupling using the Biacore amine coupling kit). After a
preconditioning step of 5 injections of RANKL008a, all samples were
diluted to 2.5 nM in triplicate and analyzed on the chip. Slopes
were determined using the general fit method and the linear fit
model (BIAevaluation software). To determine the initial binding
rate (IBR), the slope between 5 s and 30 s was selected. The values
of these slopes were transferred in excel and the percentage
activity/potency compared to the RANKL008a reference material was
determined. Biacore potency is thus expressed as relative potency
compared to the reference materials.
Example 1.2
Stability of the Nanobody in Different Buffers after Different
Freeze/Thaw Cycles
[0697] A freeze/thaw stability study was performed to determine the
effect of repetitive freeze and thawing on the recovery, physical
stability and chemical stability of RANKL008a. Aliquots of batch
RANKL008a formulated at .about.60-85 mg/mL in the buffers 1-12
given in Table 1 were subjected to 10 freeze/thaw (F/T) cycles at
-20.degree. C. One F/T cycle is defined by freezing the sample for
1 hour in a freezer at -20.degree. C. followed by thawing at room
temperature for 30 minutes. The stressed samples were compared with
reference material (stored at 4.degree. C.) using SE-HPLC (FIG. 1
(A); representative figure of the experiments performed in
phosphate buffer), RP-HPLC (FIG. 2; representative figure of the
experiments performed in phosphate buffer) and the ELISA potency
assays (Table 2). All other data of the freeze thaw experiments
demonstrate similar patterns as given in FIGS. 1 and 2 (except FIG.
1 (B) see below).
[0698] Subjecting RANKL008a to 10 F/T cycles had no significant
effect on its stability: the SE-HPLC- and RP-HPLC profiles were
comparable between the reference batches and material subjected to
multiple freeze/thaw cycles. There was no decrease in the total
surface area and no new peaks were being formed, except in 20 mM
L-histidine, pH 5.5+10% mannitol and 20 mM L-histidine, pH 6+10%
mannitol, where the main peak had a very small shoulder in the
SE-HPLC chromatograms (FIG. 1 (B) showing the data at pH 5.5 and
where the minor shoulder on the main peak is indicated by an
arrow). In Table 3 and Table 4 data are included for the
integration of the different peaks (expressed as % surface area) in
the SE-HPLC and RP-HPLC analysis respectively. These data
demonstrate that no changes occur in the profiles after 10 freeze
thaw cycles.
[0699] Analysis by the ELISA potency assays indicated no loss of
activity after repetitive freezing and thawing in all formulations,
except in 20 mM Histidine, pH 5.5+10% mannitol where there appeared
to be a lower RANKL and HSA binding potency.
Example 1.3
Stability of the Nanobody in Different Buffers when Stored at
37.degree. C. Up to 10 Weeks
[0700] RANKL008a was formulated in different buffers at
.about.60-85 mg/mL (buffers 1-12 given in Table 1). The stability
of the different samples was assessed in accelerated stress
conditions at 37.degree. C..+-.3.degree. C. Samples were taken
after 2, 3, 5 and 10 weeks storage at this temperature and were
analyzed using SE-HPLC, RP-HPLC and IEX-HPLC. Biacore was performed
on the samples stored for 10 weeks to evaluate loss in potency.
1.3.1 SE-HPLC Analysis
[0701] The results of the analysis of a sample by SE-HPLC is given
in FIG. 3 where an example is shown for the sample stored during
two weeks at 37.degree. C. in the presence of 50 or 100 mM salt or
10% mannitol phosphate buffer. Storage at 37.degree. C. resulted in
the formation of a clear prepeak eluting at about 40 minutes and
some minor postpeaks close to the main peak; at the 60 minutes
elution time (see insert in FIG. 3) some degradation fragments
could be observed. In Table 3 the integration data for all samples
analysed is summarized for the different peaks observed (except
peaks after 60 minutes elution time). The peak area of the prepeak
increased over time but was reduced by the addition of mannitol to
the buffer (Table 3). The postpeaks after 60 minutes elution time
corresponded to degradation products (due to remaining proteolytic
activity in sample). The relative area (%) of these peaks increased
only slightly, implying that degradation was restricted to a
minimum.
[0702] The prepeak represented the dimeric form of RANKL008a. The
peak surface area of the prepeak increased with storage time (Table
3) and was accompanied by a concomitant decrease in surface area of
the main peak (Table 3). The propensity to form dimers was
significantly lower in the formulations containing 10% mannitol,
which seemed to have a positive effect in suppressing the
dimerization process. Note the significant lower amounts of dimers
observed in the Acetate and Histidine buffers (pH 5.5) containing
10% mannitol (Table 1 and FIG. 4). FIG. 4(A) summarizes the %
surface area for the main peak in the different buffers and at
different time points when stored at 37.degree. C. FIG. 4(B)
summarizes the data for the % prepeak (dimer).
1.3.2 RP-HPLC Analysis
[0703] A representative RP-HPLC is given in FIG. 5 where the
RP-HPLC chromatogram of a RANKL008a sample stored in phosphate
buffer with different concentrations salt or 10% mannitol is
represented. The RP-HPLC profiles of RANKL008a formulated in the 12
different buffers were comparable to this Figure. In Table 4
integration data for the different peaks detected is summarized. In
FIG. 5 the inset shows a zoom on the main peak where the two
pre-peaks can be discriminated, while the post peak that is fully
base line resolved from the main peak is the pyro-glutamate variant
of the RANKL008a where the N-terminal glutamic acid has been
converted to the pyroglutamate form.
[0704] There were two differences between the RP-HPLC profiles of
the reference batch and the storage samples. Firstly, the
pyroglutamate peak increased with increased incubation time and was
more apparent in phosphate buffer at pH 7 than at pH 5.5 or pH 6.
Secondly, a prepeak was being formed in function of storage time.
The surface area of this peak was higher in the phosphate
buffer.
[0705] There were no differences in the RP-HPLC profiles of the
samples without or with mannitol.
1.3.3 IEX-HPLC Analysis
[0706] A representative IEX-HPLC chromatogram of the RANKL008a
stored for 2 weeks in phosphate buffer with different salt
concentrations or 10% mannitol is depicted in FIG. 6. The inset
shows a zoom in on the main peak where a minor post peak 1 and a
more significant post-peak 2 is observed. Results of the analysis
of the different samples by IEX-HPLC are given in Table 5 and FIG.
7.
[0707] The first postpeak constituted maximally 4.5% of the total
peak surface area. The surface area of this peak was the highest in
phosphate buffer and the lowest or even absent in the
mannitol-containing buffers. The peak area of the second peak on
the other hand was substantial, yet significantly lower in the
buffers containing 10% mannitol (FIG. 7). The material eluting in
the post-peak was collected by fraction collection and
re-chromatographed on the SE-HPLC column described above. This
post-peak 2 elutes in the SE-HPLC chromatogram at the dimer
position demonstrating that i) this dimer does not dissociate under
these conditions and that ii) this dimer elutes later on the
IEX-HPLC column. Therefore we can conclude that the post-peak 2 is
the dimerized form of the RANKL008a.
1.3.4 Biacore Potency Analysis of the RANKL008a Stored at
37.degree. C.
[0708] The RANKL and HSA binding of RANKL008a in stability samples
stored for 10 weeks at 37.degree. C. was compared with the activity
of the unstressed reference batch using Biacore analysis. The
relative potencies are given in Table 6 and are expressed as %
activity compared to reference batch.
[0709] After 10 weeks of storage at 37.degree. C. the relative
potency of RANKL008a for binding RANKL had dropped to 70-80% in the
different buffers (Table 6). In histidine, pH 6+10% mannitol, the
activity remained the highest (87.4%). The higher the NaCl
concentration in the buffer, the lower the relative potency in the
sample (compare the values obtained in buffers with 50 mM NaCl and
100 mM NaCl in Table 6).
[0710] The relative potency for HSA binding had dropped more
compared to the activity for RANKL binding after 10 weeks storage
at 37.degree. C. This decrease in activity however was less
significant in the mannitol-containing buffers than in the
NaCl-containing buffers. As observed for RANKL binding, the
percentage activity on HSA decreased with increasing concentrations
of NaCl in the different buffers.
Example 1.4
Osmolality Measurement for the Nanobodies in the Different
Buffers
[0711] Osmolality measurements were performed on the different
formulations used in the stability studies: [0712] RANKL008a in 10
mM Phosphate/10% mannitol: 635 mOsm/kg [0713] RANKL008a in 10 mM
Acetate/10% mannitol: 643 mOsm/kg [0714] RANKL008a in 20 mM
L-histidine pH 5.5/10% mannitol: 712 mOsm/kg [0715] RANKL008a in 20
mM L-histidine pH 6.0/10% mannitol: 667 mOsm/kg [0716] RANKL008a in
10 mM acetate buffer pH 5.5/100 mM NaCl: 272 mOsm/kg [0717]
RANKL008a in 10 mM phosphate/5% mannitol: 389 mOsm/kg
[0718] Formulations containing 10% mannitol were hypertonic.
Example 1.5
Stability of the Nanobodies During Mechanical Stress
[0719] Mechanical stress experiments were performed on RANKL008a
(62.2 mg/mL) in 10 mM phosphate buffer pH 7.0 with 115 mM NaCl. The
RANKL008a sample was diluted (in the 10 mM phosphate buffer pH 7.0
with 115 mM) or undiluted with and without 0.01% Tween 80. The
samples were shaken, stirred, rotated and pushed through a needle
with a syringe (the syringe used can be any commercially available
syringe, such as e.g. a 1 mL, 2 mL, 3 mL, 4 mL, 5 mL, 10 mL, 20 mL,
30 mL, 40 mL up to 50 mL syringe) as follows: [0720] Diluted to 5
mg/mL or undiluted and shaken (10 s-1 min); [0721] Pushed through a
syringe (3 mL) with needle 25 G (undiluted) (10.times.); [0722]
Rotated (10 rpm) on an end over end mixer for 2 days at room
temperature (undiluted) [0723] Stirred 1 hour at room temperature
for 2 days at 5.degree. C. (diluted to 5 mg/mL) The different
samples were compared visually for any differences in
appearance.
[0724] Strong shaking for a short time (10 s) caused strong foaming
of the samples in the absence of Tween 80, the diluted sample got
very opaque (FIG. 8) while this was less pronounced for the
undiluted sample. In the presence of the Tween 80 this opacity was
not observed.
[0725] The undiluted RANKL008a sample with and without 0.01% Tween
80 was pushed 10 times through a needle (25 G) with a 3 mL syringe.
The sample without Tween 80 got opaque, there was also formation of
foam and tiny air bubbles were visible when tapping the vial, in
the vial with 0.01% Tween 80 opacity was limited.
[0726] The undiluted RANKL008a sample with or without 0.01% Tween
80 was rotated for 2 days at 10 rpm. Both samples stayed clear.
[0727] The diluted (to 5 mg/mL) RANKL008a sample with or without
0.01% Tween 80 was stirred for 1 hour at room temperature and
further for 2 days at 5.degree. C. The visual observations are as
follows: stirring during 1 h at room temperature induced an opacity
which was not observed in the presence of 0.01% Tween 80. After
stirring 1 hour at room temperature, the sample without Tween 80
was slightly opaque while the sample with Tween 80 stayed clear.
After 2 days stirring at 5.degree. C., both sample were opaque but
the opacity in the sample without Tween 80 was higher.
[0728] With the addition of 0.01% Tween 80, the RANKL008a sample
was less or not opaque after mechanical stress and there was less
foam formation. This indicates that the sample is less susceptible
to denaturation at the air-water interface if Tween 80 is
added.
Example 1.6
Syringeability of the Different Nanobody Formulations
[0729] The effect of using different diluents--i.e. saline
solution, phosphate buffer without Tween 80 or phosphate buffer
with Tween 80--on content, visual appearance and potency of
RANKL008a at low concentration (0.28 mg/mL) was determined after
passage through different syringes and needles. RANKL008a was
diluted in different diluents followed by passage or 24 h storage
in syringes (Becton Dickinson) (FIG. 9). FIG. 9 contains the
legends to the different samples generated during this experiment
where the following codes are applied:
[0730] S25/0: storage at 25.degree. C. for 0 minute
[0731] S25/24: storage at 25.degree. C. for 24 h
[0732] -TW: buffer minus Tween 80
[0733] +TW: buffer+Tween 80
[0734] PLACEBO refers to the following buffer: 10 mM
Na.sub.2HPO.sub.4 pH 7.0+115 mM NaCl
[0735] TUB: sample stored in a polystyrene tube
[0736] Visual inspection and content determination of RANKL008a
after dilution in different diluents and passage/storage in
syringes is given in Table 7. Data on turbidity measurement at 320
and 350 nm are given in FIG. 10. The relative HSA and RANKL potency
of RANKL008a after dilution in different diluents and
passage/storage in syringes is shown in FIG. 11.
[0737] Passage through a syringe slightly increased turbidity when
using 10 mM Na.sub.2HPO.sub.4, 115 mM NaCl (pH 7.0), Dilution in
saline solution caused a drop in RANKL/HSA binding activity of
18-34.0%. A similar effect was observed using 10 mM
Na.sub.2HPO.sub.4, 115 mM NaCl (pH 7.0) without Tween 80, i.e. a
drop of 15-27%. In contrast, dilution in 10 mM Na.sub.2HPO.sub.4,
115 mM NaCl (pH 7.0) with Tween 80 did not appear to have a
dramatic effect confirming the beneficial role of Tween 80 in the
buffer.
Example 1.7
Stability of Nanobody Formulations During Syringe Passage with
Different Needle Size
[0738] The effect of syringe passage on visual appearance of the
RANKL008a using different needle sizes and needle size combinations
was evaluated. RANKL008a was diluted in an Eppendorf tube (TUB) in
10 mM Na.sub.2HPO.sub.4, 115 mM NaCl, 0.01% Tween 80 (v:v), (pH
7.0) to a final concentration of 0.28 mg/mL followed by single
passage through a 1 mL Becton Dickinson syringe equipped with
different needles (i.e. Terumo 18 G, 23 G, 27 G and 30 G) (FIG.
12). In this Figure and Table 8 the following codes apply:
[0739] +TW: buffer+Tween 80
[0740] PLACEBO refers to the following buffer: 10 mM
Na.sub.2HPO.sub.4 pH 7.0+115 mM NaCl
[0741] TUB: sample stored in a polystyrene tube
[0742] 18 G/18 G: sample drawn up with a 18 G needle and expelled
through a 180 needle
[0743] 18 G/27 G: drawn up with a 18 G needle and expelled through
a 27 G needle
All other coding is similar to the two examples given above.
[0744] Turbidity was determined by visual inspection and by
measurement of the absorption at wavelengths of 320 nm, 340 nm, 350
nm and/or 500 nm, and determining the ratio of the obtained value
over the absorption at A278 nm (mostly 320/278 and 350/278). A
ratio of >0.05 was considered significant. Visual inspection,
content and turbidity of RANKL008a before (TUB) and after passage
through syringes with different needle size is shown in Table
8.
[0745] In a further experiment, RANKL008a was subjected to single
passage through a 1 mL syringe equipped with different needle sizes
(i.e. 270 and 290) both undiluted (65 mg/mL) and diluted in 10 mM
Na.sub.2HPO.sub.4, 115 mM NaCl, 0.01% Tween 80 (v:v), (pH 7.0) to a
final concentration of 0.28 mg/mL (FIG. 13). Visual inspection,
content and turbidity of RANKL008a before (TUB) and after passage
through syringes with different needle size is shown in Table
9.
[0746] In Table 9 and FIG. 13 the following codes are used: [0747]
+TW: buffer+Tween 80 [0748] PLACEBO refers to the following buffer:
10 mM Na.sub.2HPO.sub.4 pH 7.0+115 mM NaCl [0749] TUB: sample
stored in a polystyrene tube [0750] 27 G/27 G: sample drawn up with
a 27 G needle and expelled through a 270 needle [0751] 29 G/29 G:
drawn up with a 290 needle and expelled through a 290 needle [0752]
T: Terumo needle, B Becton Dickinson needle [0753] 0028 refers to
concentration at 0.28 mg/mL, 6500 to about 65 mg/mL.
[0754] Different combinations of needle sizes did not have a
significant effect on RANKL008a recovery or sample turbidity both
at low (0.28 mg/mL) and high (62.8 mg/mL) concentration (up to
gauge sizes 29 and 27 respectively). In both experiments turbidity
values were low (<0.05).
Example 2
Formulation and Stability Studies with Nanobodies that Bind
IL-6R
Example 2.1
Materials and Methods Used in the Study
2.1.1 Single Variable Domains
[0755] Three Nanobodies that were used in this study have been
described in PCT application No. PCT/EP2010/054764 to Ablynx N.V.
IL6R304 is a bispecific Nanobody consisting of two humanized
variable domains of a heavy-chain llama antibody, one binding to
IL-6R, the other one (Alb8) binding to HSA. The trivalent
bispecific Nanobodies IL6R305 and IL6R306 consist of two identical
subunits that are specific for IL-6R while the third subunit binds
to HSA. The build-up of the subunits differs in IL6R305 and IL6R306
(see Table C-27 of PCT/EP2010/054764). The subunits in all three
Nanobodies are fused head to-tail with a 9G/S linker. The sequences
and characteristics of the three Nanobodies are given in Table
10.
[0756] The Nanobodies were expressed in Pichia pastoris.
Concentration of the Nanobody and buffer switch to PBS or other
formulation buffer was done via UF/DF (Sartorius Hydrosart Sartocon
Slice 200, 10 kDa). A final filtration was carried out at 0.22
.mu.m. An overview of the different IL-6R Nanobody batches is given
in Table 11.
[0757] Unstressed samples in PBS or other formulations were used as
reference material for analyzing the storage stability samples.
2.1.2 Other Reagents
[0758] Reagents used in the study are given in Table 12. The
complete composition of D-PBS was 137 mM NaCl, 2.7 mM KCl, 10 mM
Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic.
2.1.3 Equipment and Methods for Measurements
[0759] HPLC experiments were carried out on an Agilent 1200 series
instrument from Agilent Technologies (Palo Alto, USA) or on a
Dionex Ultimate 3000 instrument. The columns used were: [0760]
RP-HPLC: Zorbax 300SB-C3 5-micron, 4.6.times.150 mm (Agilent, Cat.
No. 883995-909) or Zorbax 300513-C8 5-micron, 4.6.times.150 mm
(Agilent, Cat. No. 883995-906); [0761] SE-HPLC: Phenomenex BioSep
SEC 52000 (00H-2145-KD)
[0762] Concentration determinations of the Nanobodies were done
with Nanoprop ND-1000 (Thermoscientific), with a Uvikon 943
Spectrophotometer (Kontron Instruments) or with an Eppendorf
Biophotometer 6131 at 280 nm.
[0763] Particle size distribution was measured on a PAMAS SVSS-C
particle counter (PArtikelMess-und AnalyseSysteme GMBH).
[0764] Osmolality measurement was done with an osmometer Model 3320
from Advanced instruments.
[0765] For measurement of binding activity Biacore 3000 (GE
Healthcare) was used.
[0766] The thermal shift assay (TSA) was performed on a
LightCycler480 Q-PCR device (Roche).
[0767] For determination of the Tm, an automated VP-capillary
Differential Scanning calorimeter (DSC, MicroCal) was used.
2.1.4 Purity Assay of the IL-6R Nanobodies by Size Exclusion High
Performance Liquid Chromatography (SE-HPLC)
[0768] The SE-HPLC assay consisted of a pre-packed Phenomenex
BioSep SEC 52000 column, a mobile phase consisting of KCl, NaCl and
phosphate buffer pH 7.2 (D-PBS) and UV detection at 280 nm. The
relative amount of specific protein impurity was expressed as area
%, and was calculated by dividing the peak area corresponding to
the protein impurity by the total integrated area.
[0769] The method can resolve and quantify the relative amounts of
intact material and product related impurities such as aggregates
and degradation fragments.
2.1.5 Purity Assay of the IL-6R Nanobodies by Reverses Phase High
Performance Liquid Chromatography (RP-HPLC)
[0770] In the RP-HPLC assay a Zorbax 300SB-C3 or Zorbax 300513-C8
column (Agilent Technologies, Palo Alto, US) at elevated
temperatures were used. With the C3 column, mobile phase A
consisted of 0.1% TFA and mobile phase B consisted of 0.1% TFA in
ACN/isopropanol. With the C8 column, mobile phase A consisted of
0.1% TFA and mobile phase B consisted of 0.1% TFA in 1-propanol.
The relative amount of a specific protein impurity was determined
by measuring the light absorbance (280 nm) of the components
eluting from the RP-HPLC column. The relative amount of a specific
protein impurity, expressed as area %, was calculated by dividing
the peak area corresponding to the impurity by the total integrated
area.
2.1.6 Measurement of Particle Size Distribution (PAMAS)
[0771] The measurements on the PAMAS SVSS-C particle counter were
performed as follows: 100 .mu.l sample was diluted 1/10 in 1 mL
MilliQ water and 10 consecutive measurements were performed in all
16 channels (diameter set 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25,
50, 100, 150 and 200 .mu.m). For calculation of the average value,
the first 2 measurements were excluded and the dilution factor was
taken into account. The results are given as cumulative data (total
particle counts>x .mu.m) or differential data (total particle
counts between diameter x and y .mu.m). Only the cumulative data
are presented.
2.1.7 Affinity Measurement on Biacore
[0772] A chip was first immobilized with HSA (amine coupling using
the Biacore amine coupling kit).
[0773] After a preconditioning step of 5 injections of the
Nanobody, all samples were diluted to 2.5 nM in triplicate and
analyzed on the chip. Quality control of the chips using the
reference sample was included in the experiment to detect any loss
of activity or decrease in response (deterioration of the chip).
Slopes were determined using the general fit method and the linear
fit model (BIAevaluation software). To determine the initial
binding rate (IBR), the slope between 5 s and 30 s was selected.
The values of these slopes were transferred in excel and the
percentage activity compared to the reference was determined.
2.1.8 ELISA Based Potency Assay for HSA Binding
[0774] Human Serum Albumin (HSA) was immobilized onto a multiwell
Maxisorp ELISA plate by adsorption. After blocking excess binding
sites on the plates with Superbiock T20 (PBS) blocking buffer, a
dilution series of test and reference samples was applied on the
plate. Bound Nanobody was subsequently detected using a bivalent
anti-Nanobody Nanobody directly conjugated to horseradish
peroxidase (HRP). In the presence of H.sub.2O.sub.2 HRP catalyzes a
chemical reaction with Tetramethylbenzidine (es TMB) which results
in the formation of a color. The reaction was stopped by adding 1N
HCl. The optical density of the color was measured at 450 nm.
2.1.9 ELIAS Based Potency Assay for IL-6R Binding
[0775] For the reference, control and test samples, different
dilutions of the Nanobodies were prepared. These dilutions were
pre-incubated with a constant amount of 100 ng/mL IL-6, followed by
the addition of 4 ng/mL soluble IL-6R. Subsequently, this mixture
was transferred to a microtiter plate coated with a non
neutralizing anti-IL-6R Nanobody. After washing, residual bound
IL-6 was detected with biotinylated anti-human IL-6 monoclonal
antibody, followed by HRP-labeled streptavidin detection. In the
presence of H.sub.2O.sub.2 HRP catalyzes a chemical reaction with
Tetramethylbenzidine (es TMB) which results in the formation of a
color. The reaction was stopped by adding 1N HCl. The optical
density of the color was measured at 450 nm. The relative potency
of the test samples compared to the reference sample was analyzed
by use of PLA 2.0 Software.
2.1.10 Capillary Isoelectric Focusing (cIEF)
[0776] Capillary isoelectric Focusing (cIEF) is an
analysis/separation technique that differentiates proteins with
respect to charge, i.e., it separates proteins according to their
isoelectric points (pl). The separation principle is similar to
gel-based/flatbed IEF but differs mainly in its format, that is,
the separation takes place in an open tube of narrow format
(capillary) eliminating the need for any anticonvective matrix
support. Also, cIEF is a fully automated instrument with online
detection and data acquisition. A drawback of traditional cIEF in a
conventional CE instrument is that the focused (stationary) zones
must be mobilized past the single-point detection area in order to
record the signal. During mobilization the zones may become
broadened with contaminant loss of resolution and decreased
detectability. Moreover, the analysis time and risk of protein
aggregation/precipitation will increase. By imaging cIEF the
focusing process is followed in real-time over the
whole-column/capillary by a CCD camera excluding the mobilization
step. As soon as the focusing process is completed the analysis run
is finished.
Example 2.2
Tm Determination
[0777] The melting temperature (Tm) in different buffers was
determined using the fluorescence-based thermal shift assay (TSA,
for IL6R304 and IL6R305) and by differential scanning calorimetry
(DSC, for IL6R304).
2.2.1 Thermal Shift Assay
[0778] The thermal shift assay or ISA can be performed in 96-well
plate in a Q-PCR device to evaluate the effect of buffer couple,
ionic strength, pH and excipients on the thermal stability of
proteins. The assay results in a Tm value that is indicative for
the thermal stability in the tested buffers. Briefly, the assay
follows the signal changes of a fluorescence dye, such as Sypro
Orange, while the protein undergoes thermal unfolding. When Sypro
Orange is added to a properly folded protein solution, it is
exposed in an aqueous environment and its fluorescence signal is
quenched. When the temperature rises, the protein undergoes thermal
unfolding and exposes its hydrophobic core region. Sypro Orange
then binds to the hydrophobic regions, unquenches which results in
the increase of the fluorescence signal.
[0779] In a first experiment, the Tm was assessed for IL6R304 and
IL6R305 in different buffers, excipients and combinations thereof
using the TSA assay. The obtained Tm values are displayed
graphically in FIG. 14 and FIG. 15.
[0780] In all conditions tested, the Tm values were slightly higher
for IL6R304 than for IL6R305. The buffers and excipients tested had
a similar effect on the Tm values of IL6R304 and IL6R305:
[0781] Effect of buffer pH: the highest melting points were
obtained in Hepes buffer (pH 7 and pH 8) and L-histidine pH 6.5,
followed by phosphate buffer (pH 6.7 and 7.7), Tris pH 7.2 and
succinate buffer pH 6.2. The lowest melting points were obtained in
PBS (58.82.degree. C. for IL6R304) and in the buffers with the
lowest pH, i.e. succinate pH 5.2 and L-histidine pH 5.5. The higher
melting point in L-histidine pH 6.5 correlates well with the higher
solubility of IL6R304 in this buffer (see Example 2.3).
[0782] Effect of [NaCl] concentration: the highest melting
temperatures were measured when no sodium chloride was added to the
buffers. Tm values decreased gradually with increasing NaCl
concentration; the effect plateaus at 300 mM NaCl.
[0783] Effect of the excipients mannitol, sucrose and glycine: all
excipients tested appeared to have a stabilizing effect on IL6R304
and IL6R305, since the melting temperatures increased with
increasing excipient concentration. The highest Tm values were
obtained in buffers containing 7.5% mannitol or 5% sucrose.
[0784] In summary, IL6R304 and IL6R305 seemed to be more stable in
buffers that had a neutral pH and which contained a low NaCl
concentration and significant amounts of mannitol or sucrose. This
is represented schematically in Table 13. L-histidine pH 6.5
appeared to be a good buffer to take forward in further stability
testing and formulation work. Another important argument for
proceeding with a L-histidine buffer is that the solubility of
IL6R304 was shown to increase dramatically in this buffer compared
to PBS (see Example 2.3). Buffers containing high NaCl
concentration should be avoided, while Hepes buffers at neutral pH
could preferably be used.
2.2.2 Differential Scanning Calorimetry
[0785] To identify other suitable buffers to be used in
purification protocols, i.e. in which the protein displays
acceptable stability, differential scanning calorimetry (DSC) was
used to determine the melting temperature of IL6R304 in different
candidate buffers.
[0786] FIG. 16 shows the results of a DSC experiment performed on
IL6R304 formulated in different buffers. The highest melting
temperatures were observed in MES (pH 6.0) and Hepes (pH 7.0),
whereas acetate, Tris-HCl and phosphate indicate a slightly lower
thermal stability. The Tm value obtained in citrate (pH 3.5) was on
average 10.degree. C. lower than in the other buffers. All heat
capacity (Cp) melting peaks were sharp and rather symmetrical in
all buffers. The restoration of a base-line after the transition
indicated that no precipitation had occurred.
[0787] An overview of all Tm values obtained in the DSC experiments
is shown in Table 14. From these results we can conclude that
adding more NaCl lead to a gradual decrease in melting temperature,
as was also observed in the ISA.
2.2.3 TSA Experiment Using Experimental Design
[0788] A second TSA experiment was performed on IL6R304 using
experimental design (DOE). The DOE consisted of different steps.
The outcome of each step was used to define the DOE for the next
step. Briefly, the experimental set-ups and obtained results were
the following: [0789] Step 1: L-histidine, succinate, phosphate and
Tris were tested at different pH (6-7) and buffer strength (10-40
mM). The most promising formulation was found to be low ionic
strength L-histidine and Phosphate buffers with a pH of 7 and 6,
respectively. [0790] Step 2: the effect of adding NaCl and
different excipients to 15 mM L-histidine pH 6.5 and 7 and 15 mM
Phosphate pH 6 and 6.5 was tested. One representative of three
excipient families was included: mannitol (polyol), sucrose
(non-reducing sugar) and arginine (amino acid). It was concluded
that Arginine and/or NaCl decrease the Tm of IL6R304. A maximal
melting temperature was reached in sucrose or a mix of
sucrose/mannitol (10% in total). Furthermore, a higher Tm was
obtained in L-histidine than in phosphate. For both buffers, best
results were obtained at pH 6.5. [0791] Step 3: other
representatives, i.e. sorbitol, xylitol, ribitol, trehalose and
glycine, from the three excipient families were tested in 15 mM
L-histidine and 15 mM phosphate, both at pH 6.5. Again, the Tm of
IL6R304 was higher in L-histidine buffer than in phosphate for all
excipients and excipient combinations. The best buffer formulation
contained 10% sorbitol or trehalose. (Table 15)
[0792] In a fourth step, the outcome of the statistical analysis of
the DOE was confirmed by a repeat of the TSA and by differential
scanning calorimetry.
Example 2.3
Solubility of the Nanobodies in Different Buffers
2.3.1 Solubility in PBS and the Effect of Tween 80
[0793] During downstream processing and storage of the IL6R304,
IL6R305 and IL6R306Nanobodies in D-PBS buffer, precipitation
occurred. Precipitates were already formed during storage overnight
at 5.degree. C. or -20.degree. C., even in samples that were
filtered (0.22 .mu.m) before storage. This not only resulted in
significant product loss, but also made it inconvenient to perform
subsequent experiments since filtration steps needed to be
incorporated constantly. From these observations it was clear that
PBS is not a suitable formulation buffer for any of the IL-6R
Nanobodies and that alternative storage buffers needed to be
identified. In an initial experiment, it was assessed whether Tween
80 could prevent this precipitation (aggregation) from
occurring.
[0794] Briefly, IL6R304 (P#051108nr1) was diluted to 2 mg/mL in PBS
buffer, PBS buffer+0.1% (v:v) Tween 80 or PBS buffer+0.2% (v:v)
Tween 80. The three samples were stored for 4 days at 5.degree. C.
and subsequently analyzed for visible particulates (appearance
testing by visual inspection; Table 16), sub-visible particle
counts (PAMAS; FIG. 17), via UV spectroscopy (A320/A280 ratio, i.e.
measure for the presence of particulates; Table 16) and SE-HPLC
(FIG. 18), Significantly more and larger particles were present in
the IL6R304 sample formulated in PBS compared to the IL6R304 sample
formulated in PBS+Tween 80.
2.3.2 Concentration Experiments to Determine the Solubility of
IL6R304 and IL6R305
[0795] IL6R304 and IL6R305, both formulated in PBS, were
concentrated stepwise with a Vivascience concentrator (Vivaspin 500
5,000 MWCO, 500 .mu.l concentrator). During the concentration
experiment, the retentate was mixed gently regularly and was
analyzed visually for particulates/precipitation. The presence of
insoluble aggregates in the retentate was verified by checking the
protein concentration before and after centrifugation at maximum
speed. Based on the results from the thermal shift assay (see
Example 2.2.1), the solubility of IL6R304 was also analyzed in 20
mM Histidine, pH 6.5.
[0796] In conclusion, the solubility of IL6R304 and IL6R305 in PBS
was limited, with estimated values of 20 mg/mL, and 15 mg/mL,
respectively (concentration at which protein precipitation
occurred). No precipitation of IL6R304 was observed at a
concentration between 20-90 mg/mL in L-histidine buffer suggesting
that the solubility can be increased significantly by changing the
formulation buffer. Note that no significant protein loss has been
observed.
2.3.3 Determination of the Theoretical Solubility
[0797] The theoretical solubility of IL6R304 and IL6R305 was
determined using the PEG exclusion method in PBS buffer (IL6R304,
IL6R305) or in 20 mM L-histidine, pH 6.5 (IL6R304). Briefly, a
concentrated Nanobody solution (30-80 mg/mL) in the respective
buffer was incubated for 15 minutes at room temperature in the
presence of increasing concentrations of PEG6000. After
centrifugation at 20000.times.g for 3 minutes, log values of the
[soluble protein concentration] were plotted versus PEG6000
concentration (FIG. 19). By regression analysis and extrapolation
to a zero concentration of PEG6000, the theoretical maximum protein
concentration (and thus solubility values) could be obtained for
the Nanobodies in the buffers tested. The obtained solubility
values correlated well with the values obtained experimentally
using the stepwise concentration experiments with the Vivaspin
centrifugal concentrators. When comparing the solubility of IL6R304
in PBS and L-histidine pH 6.5, it could be concluded that the
solubility of IL6R304 increased significantly in the L-histidine
buffer.
Example 2.4
Storage Stability Study of the Nanobodies at 37.degree. C.
[0798] An initial storage stability study was performed to get a
general understanding of the stability of the IL-6R Nanobodies and
to determine if adding mannitol in the formulation buffer has a
beneficial effect in minimizing the formation of potential dimers,
as was observed for RANKL008a (see Example 1.3).
[0799] The three IL-6R Nanobodies were formulated in different
buffers (Table 17) at a concentration of 10 mg/mL (IL6R304), 7.1
mg/mL (IL6R305) and 10.3 mg/mL (IL6R306). The stability of the
different samples was assessed in accelerated stress conditions at
37.degree. C. Samples were analyzed after 1 week using SE-HPLC and
RP-HPLC. Selected samples of IL6R304 and IL6R305 were also analyzed
after 3 weeks of storage.
2.4.1 SE-HPLC Analysis
[0800] For all three IL-6R Nanobodies, prolonged storage at
37.degree. C. resulted in the formation of prepeaks and some minor
postpeaks. The postpeaks probably corresponded to degradation
products (due to remaining proteolytic activity in sample). The
surface area of these postpeaks remained very low, suggesting only
minimal degradation after 3 weeks at 37.degree. C.
[0801] All three IL-6R Nanobodies had a strong tendency to form
dimers/oligomers (aggregates), which were visible as prepeak(s) in
the chromatograms of the SE-HPLC analysis. An example chromatogram
is shown in FIG. 20. The peak area of the prepeak increased
significantly over time (represented as % aggregates in FIG. 21 and
FIG. 22) and was accompanied by a concomitant decrease in surface
area of the main peak. The propensity to form dimmers/oligomers
appeared to be somewhat higher for IL6R305 than for IL6R304. Also,
more dimmers/oligomers were being formed in PBS compared to the
other buffers that were tested. Importantly, the lowest amounts of
oligomers were observed in the mannitol-containing
formulations.
2.4.2 RP-HPLC Analysis
[0802] The RP-HPLC profile of IL6R304 at time point 0 weeks
included a main peak with a shoulder eluting before the main
material and a postpeak that was not well resolved from the main
peak. This postpeak most likely corresponded to the
pyroglutamate-containing variant of IL6R304. The surface area of
this peak increased with storage time and was highest in PBS and
phosphate buffer compared to the acetate and histidine buffers.
After 3 weeks of storage, the surface areas of two thus far
unidentified prepeaks increased (FIG. 23).
[0803] The RP-HPLC profile of IL6R305 at time point 0 weeks
included a main peak with some minor prepeaks. The resolving power
of the RP-HPLC method used was insufficient to separate the
pyroglutamate-containing variant from the main material.
Example 2.5
Osmolality Measurement
[0804] According to the European Pharmacopoeia, a solution is
considered isotonic if it has an osmolality of 290.+-.30 mOsm/kg.
Osmolality measurements on 20 mM L-histidine pH 6.5 containing
different concentrations of excipients were therefore performed to
define the range of excipient concentration that would be
acceptable for an isotonic liquid formulation of IL6R304.
[0805] IL6R304 (10 mg/mL) was formulated in the different buffers
of Table 18. The results from osmolality measurement of these
formulations are shown in FIG. 24.
Example 2.6
Storage Stability Study of IL6R304 at 5.degree. C. and 37.degree.
C.
[0806] An overview of the different formulation buffers and methods
used in stability testing of IL6R304 batch P#051108nr1 is given in
Table 18 and Table 19, respectively. Because IL6R304 was found to
be prone to aggregation and precipitation, Tween 80 was added to
most formulations.
2.6.1 Appearance and OD280
[0807] No turbidity was observed in the samples stored for 5 weeks
at 5.degree. C., indicating that 20 mM L-histidine pH 6.5 is a much
better storage buffer for IL6R304 than PBS.
[0808] After storage for 1 week at 37.degree. C., a slight
turbidity was observed in all 12 samples. In the IL6R304 samples in
buffers 1, 2, 3, 7, 8 and 9 more opalescence was observed compared
to IL6R304 in the buffers 4, 5, 6, 10, 11 and 12, which all
contained mannitol. After storage for 2 weeks at 37.degree. C.,
slightly more turbidity was observed compared to the 1 week
samples. However, the trend observed for the 1 week samples
continued: the IL6R304 samples in buffers 1, 2, 3, 7, 8 and 9
showed more opalescence compared to the mannitol-containing
buffers. After storage for 5 weeks at 37.degree. C., turbidity was
still present and slightly less in the samples containing mannitol.
However, it seemed like the turbidity had not increased compared to
the 2 weeks samples.
[0809] Despite the opacity observed in the stressed samples, the
protein concentration in the samples has not decreased
significantly (data not shown) although there was a slight trend to
a lower concentration due to a higher turbidity. Also, the
OD320/OD280 ratio, which is a measure for turbidity or the presence
of particulates, was <0.05 in all buffer conditions. In fact,
the ratio was 2-10 fold lower than observed in the unstressed
sample in PBS, again showing that the L-histidine pH 6.5 buffer has
a stabilizing effect on IL6R304.
2.6.2 SE-HPLC Analysis
[0810] Samples of the reference material (0 weeks) and samples
stored for up to 6 months at 5.degree. C. and 37.degree. C. were
analyzed using SE-HPLC.
[0811] No differences were observed between the SE-HPLC profiles of
the reference samples (at 0 weeks) and the samples stored for up to
5 weeks at 5.degree. C. In addition, there were no significant
differences between the different buffers. The small amounts of
aggregates already present in the start material were not
increasing with prolonged storage time (FIG. 25(B)), indicating
that the Histidine buffer had a stabilizing effect on ILR304, even
in the absence of excipients such as Tween 80, mannitol or sucrose.
Note that IL6R304 formed aggregates when stored for a short time
(hours-days) at 5.degree. C. in D-PBS buffer.
[0812] SE-HPLC analysis of the samples stored for 6 months at
5.degree. C. also did not show increase in area % of the prepeaks,
meaning that no oligomers were formed under these storage
conditions, not even in the formulation containing only 20 mM
L-histidine, pH 6.5 i.e. without Tween-80 or any excipient (data
not shown).
[0813] Prolonged storage at 37.degree. C. resulted in the formation
of prepeaks and some minor postpeaks. The postpeaks probably
corresponded to degradation products (due to remaining proteolytic
activity in sample). The relative area (%) of these peaks increased
only slightly, implying that degradation was restricted to a
minimum. The other peaks visible in the chromatograms were
background peaks arising from the buffer components.
[0814] The peak area of the prepeaks increased significantly over
time (FIG. 25 (A) and FIG. 26 (B)). Given the relative position of
the prepeaks to the main peak, the prepeaks most likely represented
dimeric or oligomeric forms (aggregates) of IL6R304. The peak
surface area of the prepeak increased with storage time and was
accompanied by a concomitant decrease in surface area of the main
peak.
[0815] An important observation was that the propensity to form
dimers/oligomers was buffer-dependent: the propensity to
oligomerize was significantly lower in the mannitol- and
sucrose-containing formulations. Glycine appeared not to have such
a positive effect in preventing the oligomerization process. Tween
80 had no inhibitory effect on the formation of oligomers.
[0816] Importantly, the % oligomers observed in all 12 L-histidine
buffers after storage for 3 weeks at 37.degree. C. was
significantly lower than the equivalent sample in D-PBS buffer,
i.e. 2.2-4.6% in L-histidine, pH 6.5 compared to 11.7% PBS (FIG.
26(A)). This buffer-dependent effect on the physical stability of
IL6R304 correlated very well with the buffer-dependent differences
observed in thermal stability testing of IL6R304 (Example 2.2): the
melting temperature of IL6R304 was found to be only 58.8.degree. C.
in PBS but is 62.8.degree. C. in 20 mM L-histidine, pH 6.5.
Increasing the intrinsic stability of IL6R304 by changing the
formulation buffer from PBS to L-histidine proved to have a clear
beneficial effect on its stability upon storage.
[0817] In the samples stored for 6 months at 37.degree. C., the
lowest % of oligomers was found in the formulation containing 10%
sucrose, again corroborating the stabilizing effect of sucrose on
IL6R304 (Table 20).
2.6.3 RP-HPLC Analysis
[0818] Samples of the reference material (0 weeks) and samples
stored for up to 5 weeks at 5.degree. C. and 37.degree. C. were
analyzed using RP-HPLC.
[0819] The RP-HPLC profiles at time point 0 weeks included a main
peak, two pre-peaks and a badly resolved postpeak. This post-peak
most probably corresponded to the pyroglutamate-containing variant
of IL6R304.
[0820] The RP-HPLC profiles of the reference batch and the
stability samples stored for up to 5 weeks at 5.degree. C. were
found to be comparable.
[0821] In the stability samples stored at 37.degree. C., the peak
surface area of the pyroglutamate peak increased with storage time
while the surface area of the main peak decreased. The total area
remained unchanged.
[0822] After 5 weeks of storage at 37.degree. C., the surface area
of the two prepeaks had increased in all buffer conditions. The
identity of these variants is unknown at the moment, but could
correspond to degradation fragments.
[0823] There were no significant buffer-dependent differences in
the RP-HPLC profiles of the different samples suggesting that
chemical modifications, such as pyroglutamate formation and
oxidation which are typically detected by RP-HPLC, were limited and
at present unaffected by the buffer.
Example 2.7
Storage Stability Study of IL6R304 at -70.degree. C., -20.degree.
C., 5.degree. C., 25.degree. C. and 37.degree. C.
[0824] IL6R304 was formulated at 10 mg/mL in the 10 different
buffers shown in Table 21, stored at -70.degree. C., -20.degree.
C., +5.degree. C. and +37.degree. C. for 8 weeks and for 1 week
+25.degree. C. Stability samples were analyzed using SE-HPLC,
RP-HPLC, 00280 and visual inspection. Selected samples were also
analyzed using Biacore (HSA binding) and potency assays (HSA and
IL-6R).
2.7.1. Storage for 8 Weeks at -70.degree. C., -20.degree. C.,
5.degree. C. and 1 Week at 25.degree. C.
[0825] IL6R304 was shown to be stable after storage for 8 weeks at
-70.degree. C., -20.degree. C., 5.degree. C. and for 1 week at
25.degree. C. in all 10 buffers tested. No significant differences
were observed in potency, turbidity, SE-HPLC and RP-HPLC profiles
between the reference material and the 10 different storage
samples.
2.7.2. Storage for 8 Weeks at 37.degree. C.
Appearance and OD280
[0826] Compared to the samples stored at -70.degree. C.,
-20.degree. C. and 5.degree. C., turbidity was observed in the
samples stored at 37.degree. C. The absorbance values at 350 nm had
increased accordingly to >0.01 AU in most buffers, although the
A350/A280 ratio was still <0.05 in all buffer conditions.
Despite the opacity observed in the stressed samples, the protein
concentration in the samples had not decreased significantly.
SE-HPLC
[0827] Prolonged storage at 37.degree. C. resulted in the
time-dependent formation of a postpeak and prepeak. The postpeak
has a retention time between 22 and 23 minutes and most likely
corresponded to IL6R304 degradation fragments. The surface area of
this peak however remained low (approximately 2%), suggesting only
minimal degradation after 8 weeks at 37.degree. C. The other
postpeaks visible in the chromatograms were background peaks
arising from the buffer components.
[0828] The SE-HPLC profile of IL6R304 at time point 0 weeks
included a main peak and two minor prepeaks, which were not
completely baseline-resolved. The surface area of the prepeaks
increased over time and was accompanied by a concomitant decrease
in surface area of the main peak. Given the relative position and
heterogeneity of the prepeaks, they most likely represented dimeric
and/or oligomeric forms of IL6R304. Because of this heterogeneity
and the decreasing resolution between the prepeaks over time, the
peaks were for simplicity integrated as a single peak.
[0829] An important observation was that the propensity to form
dimers/oligomers was buffer-dependent: about 2-fold less oligomers
were being formed in L-histidine buffer compared to phosphate
buffer (FIG. 27, FIG. 28). The lowest amount of oligomers was
observed in the trehalose-containing formulation, followed by the
sucrose-containing formulation. Overall, after storage of IL6R304
at 37.degree. C. for several weeks, the amount of oligomers present
in these buffers was significantly less than observed previously in
D-PBS or in L-histidine pH 6.5 devoid of any excipient.
[0830] The presence of a non-reducing sugar suppressed the extent
of IL6R304 oligomerization considerably.
RP-HPLC
[0831] The RP-HPLC chromatograms from the ILR304 stability samples
stored for up to 8 weeks at 37.degree. C. are shown in FIG. 29.
[0832] The RP-HPLC profile of ILR304 at time point 0 weeks included
a main peak, with 2 badly resolved shoulders eluting before the
main material, a first postpeak corresponding to the
pyroglutamate-containing variant of ILR304 and a second postpeak,
corresponding to the ILR304 variant missing one disulphide bridge.
The identity of both variants has been confirmed by LC-MS.
[0833] After storage during 1 week, the surface area of the second
postpeak had decreased to a relative area % of 0, most likely due
to spontaneous oxidation into the correctly folded molecule.
[0834] The surface area of the pyroglutamate peak increased with
storage time while the surface area of the main peak decreased. The
total surface area was not changing significantly over time or
among the different buffers. FIG. 30 clearly demonstrates that the
kinetics of pyroglutamate formation was different in L-histidine,
pH 6.5 versus phosphate, pH 6.5. At all time points, less
pyroglutamate was present in the L-histidine buffer. On the other
hand, there was no correlation between the type of excipient
present in the buffer and the amount of pyroglutamate being
observed.
[0835] After storage for 8 weeks, two new postpeaks were being
formed. The first postpeak was situated between the main peak and
the pyroglutamate peak, while the second postpeak eluted just after
the pyroglutamate peak. The identity of these variants is currently
not known.
Capillary Isoelectric Focusing (cIEF)
[0836] cIEF integration data of IL6R304 stored for 8 weeks at
37.degree. C. in the different buffers are shown in Table 22. The
samples formulated in 15 mM L-histidine, pH 6.5 contain less charge
variants compared to the phosphate buffer.
Potency Assay and Biacore
[0837] The potency of the samples stored for 8 weeks at 37.degree.
C. in buffers 1-5 was determined relative to an unstressed
reference batch using the HSA-binding ELISA and the inhibition
ELISA for IL-6R as described in Examples 2.1.8 and 2.1.9 (Table
23). The HSA binding functionality of the samples stored in buffers
1-10 was also analyzed using Biacore (Table 24). Samples formulated
in the same buffers and stored at -70.degree. C. were included as
the reference molecules.
[0838] Whereas the potency assays showed comparable HSA and IL-6R
binding potencies between the stability samples and the reference
material, Biacore analysis demonstrated some differences in HSA
binding activities.
[0839] Overall, the activities of the samples formulated in
phosphate buffer (buffers 6-10) were lower than in L-histidine
(buffers 1-5). A functionality loss of approximately 16% was
observed in the buffers containing a combination of sucrose and
glycine (buffer 4 and 9). The combination of sucrose and mannitol
(buffer 5 and 10) showed no loss of functionality of IL6R304 in the
L-histidine buffer, while a decrease of 10% was observed in the
phosphate buffer. Formulations containing either mannitol, sucrose
or trehalose showed an activity between 90 and 100% after storage
for 8 weeks at 37.degree. C.
General Conclusion about the Storage Stability Study at 37.degree.
C.
[0840] Storage for up to 8 weeks of IL6R304 in different
formulation buffers under temperature stress conditions (37.degree.
C.) resulted in the following observations: [0841] The propensity
of IL6R304 to form oligomers was dependent on the buffer and
excipient: about 2-fold less oligomers were being formed in
L-histidine buffer compared to phosphate buffer, while the presence
of a non-reducing sugar suppressed the extent of IL6R304
oligomerization even further; [0842] The chemical stability of
IL6R304 was better in L-histidine buffer compared to phosphate
buffer; [0843] The HSA binding activity was maintained longer in
L-histidine buffer compared to phosphate buffer.
Example 2.8
Stability Under Stir Stress
[0844] IL6R304 was formulated at 1 mg/mL in the 10 different
buffers shown in Table 21. Aliquots of 5 mL were stirred at maximum
speed for up to 24 hours at 2-8.degree. C. Samples were analyzed
after 2, 4 and 24 hours of stirring.
[0845] All solutions remained clear after 2 hours of stirring
(Table 25). An increase in turbidity was observed in six out of ten
buffers after 4 hours. The highest opalescence was present in
buffer 10. Overall, the increase in turbidity was more pronounced
in the phosphate buffers (buffer 6-10). These observations were
confirmed after determination of the aggregation index, defined as
100*OD350/(OD280-OD350) (FIG. 31). No soluble aggregates were
observed during SE-HPLC of the different samples.
[0846] In conclusion, the stir stress data suggest a somewhat
better stir stress stability of IL6R304 in L-histidine, pH 6.5
compared to phosphate buffer, pH 6.5. No significant differences
were observed between the various excipients, although a slightly
higher turbidity was observed in the presence of 10% trehalose and
2.5% mannitol/5% sucrose.
Example 2.9
Long-Term Stability Study at -70.degree. C., +5.degree. C. and
+25.degree. C.
[0847] IL6R304 batch CMC-D-0048, formulated in 15 mM L-Histidine,
8% sucrose, 0.01% Tween80 (pH6.5) at 10.52 mg/mL, was stored for 6
months at -70.degree. C., +5.degree. C. and +25.degree. C. Samples
were analysed after 3 and 6 months of storage by visual inspection
(appearance), A280 (content), SEC-HPLC, cIEF, RP-HPLC and potency
assays (IL6R inhibition assay and HSA binding assay). The results
are summarized in Table 42, Table 43 and Table 44 for storage at
-70.degree. C., +5.degree. C., and +25.degree. C.,
respectively.
[0848] There were no significant changes in appearance, content,
potency, cIEF and HPLC profiles between the control sample
(timepoint 0 months) and all test samples stored at -70.degree. C.
or 5.degree. C. indicating that IL6R304 is stable for at least 6
months under these conditions. Regarding the sample stored at
+25.degree. C., the following observations were made when comparing
the results of the stressed samples and the control sample: [0849]
SE-HPLC: there is a small, yet gradual increase in the surface area
of the pre peak (oligomers) and post peak (degradation fragments).
[0850] cIEF: a post peak is being formed which is believed to
correspond to the pyroglutamate variant. [0851] RP-HPLC: three new
peaks are being formed, i.e. a pre peak, most likely corresponding
to degradation fragments that are present in the samples (see also
SEC-HPLC data) and two yet unidentified post peaks. The surface
area of pre peak 2 and post peak 2 (pyroglutamate) are gradually
increasing with prolonged incubation time. [0852] No potency loss
is observed in the samples stored for up to 6 months at +25.degree.
C.
Example 3
Formulation and Stability Studies with Nanobodies that Bind
IL23
Example 3.1
Materials and Methods Used in the Study
3.1.1 Single Variable Domains
[0853] 23IL0064 (SEQ ID NO: 5;
EVQLLESGGGLVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKG
RELVATINSGSRTYYADSVKGRFTISRDNSKKTLYLQMNSLRPEDTAVYYCQTSGSGSPNFWGQGTLVTVSSG-
GGGS
GGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVK-
GRFTISR
DNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQP-
GGSLRLSCAAS
GRTLSSYAMGWFRQAPGKGREFVSRISQGGTAIYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAKD-
PS PYYRGSAYLLSGSYDSWGQGTLVTVSS) has been described as SEQ ID NO:
2616 in WO 2009/068627. 23IL0064 consists of three humanized
variable domains of a heavy-chain llama antibody: 119A3v16 and
81A12v4, binding different epitopes of IL23 p19, and the ALB8
binding HSA. 23IL0075 (SEQ ID NO: 6;
EVQLLESGGGLVQPGGSLRLSCAASGRIFSLPASGNIFNLLTIAWYRQAPGKGRELVATINSGSRTYYADSVK
GRFTISRDNSKKTLYLQMNSLRPEDTAVYYCQTSGSGSPNFWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLV-
QP
GNSLRLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNS-
LRPED
TAVYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLLESGGGLVQPGGSLRLSCAASGRTLSSYAM-
GWFRQAPG
KGREFVARISQGGTAIYYADSVKGRFTISRDNSKNTLYLQMNSLRPEDTAVYYCAKDPSPYYRGS-
AYLLSGSYDSWG QGTLVTVSS) has been described as SEQ ID NO: 2622 in WO
2009/068627. 23IL0075 consists of three humanized variable domains
of a heavy-chain llama antibody: 119A3v16 and 81A12v5, binding
different epitopes of IL23 p19, and the ALB8 binding HSA. The
subunits in both Nanobodies are fused head-to-tail with a 9G/S
linker.
[0854] 23IL0064 (3.79 mg/mL in D-PBS) and 23IL0075 (4.21 mg/mL in
D-PBS) were expressed in Pichia pastoris. After clarifying the
fermentation broth via a centrifugation step, followed by a TFF
step, the Nanobodies were captured on MabCapture A (Poros),
followed by an elution at pH 2.6 using 100 mM glycine. A buffer
switch to 1/10 D-PBS was performed, and the Nanobodies were further
polished on Poros 50HS (Poros). Finally, a treatment with 50 mM OGP
for LPS-removal was performed, followed by a final size exclusion
step using Superdex 75 pg (GE Healthcare).
[0855] Unstressed samples in D-PBS or other formulations were used
as reference material for analyzing the storage stability
samples.
3.1.2 Other Critical Reagents
[0856] Reagents used in the study are given in Table 26. The
complete composition of D-PBS was 137 mM NaCl, 2.7 mM KCl, 10 mM
Sodium Phosphate dibasic, 2 mM Potassium Phosphate monobasic.
3.1.3 Equipment and Methods for Measurements
[0857] HPLC experiments were carried out on an Agilent 1200 series
instrument from Agilent Technologies (Palo Alto, USA). The columns
used were: [0858] RP-HPLC: Zorbax 300SB-C3 5-micron, 4.6.times.150
mm (Agilent, Cat. No. 883995-909) and Zorbax 300SB-C8 5-micron,
4.6.times.150 mm (Agilent, Cat. No. 883995-906) [0859] SE-HPLC:
TSKgel G2000SW.sub.XL (Tosoh Bioscience, Japan; Part#08540) [0860]
IEX-HPLC: ProPac WCX-10, 4.times.250 mm, 10 .mu.m (Dionex)
[0861] Concentration determinations of the Nanobodies was done with
Nanoprop ND-1000 (Thermoscientific), with a Uvikon 943
Spectrophotometer (Kontron Instruments) or an Eppendorf
Biophotometer 6131 at 280 nm.
[0862] Particle size distribution was measured on a PAMAS SVSS-C
particle counter (PArtikelMess-und AnalyseSysteme GMBH).
[0863] Osmolality measurement was done with an osmometer Model 3320
from Advanced instruments.
[0864] The thermal shift assay was performed on a LightCycler480
Q-PCR device (Roche).
[0865] For determination of the Tm, an automated VP-capillary
Differential Scanning calorimeter (DSC, MicroCal) was used.
[0866] Elastic light scattering was measured in a Jasco
Spectrofluorometer (FP-6500).
3.1.4 Purity Assay of the Nanobodies by Size Exclusion High
Performance Liquid Chromatography (SE-HPLC)
[0867] The SE-HPLC assay consisted of a pre-packed silica gel
TSKgel G2000SW.sub.XL column equipped with a guard column
pre-column filter, a mobile phase consisting of KCl, NaCl and
phosphate buffer pH 7.2 (D-PBS) and UV detection at 280 nm. The
relative amount of specific protein impurity was expressed as area
%, and was calculated by dividing the peak area corresponding to
the specific protein impurity by the total integrated area.
3.1.5 Purity Assay of the Nanobodies by Reverses Phase High
Performance Liquid Chromatography (RP-HPLC)
[0868] In the RP-HPLC assay a Zorbax 300SB-C3 column or Zorbax
300SB-C8 column (Agilent Technologies, Palo Alto, US) was used. The
relative amount of a specific protein impurity was determined by
measuring the light absorbance of the components eluting from the
RP-HPLC column. The relative amount of a specific protein impurity,
expressed as area %, was calculated by dividing the peak area
corresponding to the impurity by the total integrated area.
3.1.6 Purity Assay of the Nanobodies by Ion Exchange High
Performance Liquid Chromatography (IEX-HPLC)
[0869] The IEX-HPLC assay combined the use of a pre-packed Dionex
ProPac WCX-10 weak cation exchange column, a mobile phase
consisting of citrate buffer pH5.5 and UV detection at 280 nm.
After loading the protein(s) on the column, bound materials were
eluted by a sodium chloride gradient. The relative amount of the
specific protein, variant, or impurities expressed as area %, was
calculated by dividing the peak area corresponding to the specific
protein or to any protein impurity by the total area of all
integrated peaks.
3.1.7 Measurement of Particle Size Distribution (PAMAS)
[0870] The measurements on the PAMAS SVSS-C particle counter were
performed as follows: 100 .mu.l sample was diluted 1/10 in 1 mL
MilliQ water and 10 consecutive measurements were performed in all
16 channels (diameter set 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 25,
50, 100, 150 and 200 .mu.m). For calculation of the average value,
the first 2 measurements were excluded and the dilution factor was
taken into account. The results are given as cumulative data (total
particle counts>x .mu.m) or differential data (total particle
counts between diameter x and y .mu.m). Only the cumulative data
are presented.
3.1.8 Capillary Isoelectric Focusing (cIEF)
[0871] Capillary Isoelectric Focusing (cIEF) is an
analysis/separation technique that differentiates proteins with
respect to charge, i.e., it separates proteins according to their
isoelectric points (pl). The separation principle is similar to
gel-based/flatbed IEF but differs mainly in its format, that is,
the separation takes place in an open tube of narrow format
(capillary) eliminating the need for any anticonvective matrix
support. Also, cIEF is a fully automated instrument with online
detection and data acquisition. A drawback of traditional cIEF in a
conventional CE instrument is that the focused (stationary) zones
must be mobilized past the single-point detection area in order to
record the signal. During mobilization the zones may become
broadened with contaminant loss of resolution and decreased
detectability. Moreover, the analysis time and risk of protein
aggregation/precipitation will increase. By imaging cIEF the
focusing process is followed in real-time over the
whole-column/capillary by a CCD camera excluding the mobilization
step. As soon as the focusing process is completed the analysis run
is finished.
Example 3.2
Melting Temperature of the Nanobodies
[0872] The measurement of the melting temperature of a protein in
different buffers is a faster way to screen for buffers in which
the protein has the highest physical stability. It is generally
accepted that this will be predictive for the long term stability
at lower temperatures. The melting temperature can be determined
using many different techniques.
3.2.1 Melting Temperature of 23IL0064 in Different Formulation
Buffers Measured by Thermal Shift Assay
[0873] We used the thermal shift assay (TSA), which measures the
change in fluorescence intensity when the Sypro orange binds the
hydrophobic parts of the protein that is undergoing thermal
unfolding. The thermal shift assay was performed on the
LightCycler480 Q-PCR device (Roche) making use of 96-well
plates.
[0874] In a first study, the effect of buffer couple, ionic
strength and pH on the thermodynamic stability of 23IL0064 was
evaluated in a thermal shift analysis experiment. The tested
buffers used are presented in Table 28. Each buffer was tested at a
pH interval spanning 1 pH unit around their pKa. An overview of the
results is presented in FIG. 32. The melting temperatures decreased
with increasing salt concentration. Further pH 5.2 and pH 5.5
seemed less favorable compared to pH 6.2 and above. The best
buffers were Hepes (pH 7 and 8) and Histidine pH 6.5. In all
buffers, the addition of mannitol slightly increased the melting
temperatures.
3.2.2 Melting Temperature of 231IL0064 and 23IL0075 in Different
Formulation Buffers Measured by Thermal Shift Assay and
Differential Scanning Calorimetry (DSC)
[0875] 23IL0064 and 23IL0075 were analyzed in parallel in different
formulation and purification buffers by the thermal shift assay
(TSA) (Tables 34) and DSC (Table 33). For both molecules and in all
tested buffers (acetate, MES, Hepes, TRIS, and Histidine), the
melting temperatures decreased with increasing NaCl concentration,
and the melting temperatures were highest in the presence of
mannitol.
3.2.3 Melting Temperature of 23IL0075 in a Wide Range of
Formulation Buffers Measured by Thermal Shift Assay
[0876] The melting temperature for 23IL0075 in another range of
buffers and a lower pH range was screened by ISA. For this, a
design of experiments was set up to investigate the Tm of 231IL0075
varying the following parameters: pH range between 5 and 6, buffer
concentration between 10 and 50 mM, and 4 different buffers:
histidine, acetate, phosphate and succinate. A full factorial
design+central composite design was performed, in total 22
different buffers were tested in 84 experiments. The experiments
were divided over two 96-well plates, on the second plate the pH
range was expanded to 4.8 and 6.2, and the buffer concentration to
7 and 65 mM. The phosphate buffer was only tested at pH 6 and 6.2.
From the experimental results, for each buffer a model was built
that was used to predict Tm's as a function of the buffer
concentration and the pH. The results are shown in Table 35.
[0877] Succinate clearly had overall the lowest predicted Tm
values. For acetate and histidine at low concentrations and high pH
the largest predicted Tm values were obtained. In fact the model
showed for all buffers highest predicted Tm values for lowest
buffer concentrations. According to the Design-Expert Numerical
Optimization of the model the best buffer was histidine buffer pH
6.2 (desirability 1), followed closely by acetate pH 6
(desirability 0.96) and phosphate pH 6 (desirability 0.90).
3.2.4 Confirmation of Tm by Differential Scanning Calorimetry
[0878] We confirmed the Tm determination by differential scanning
calorimetry in the buffers selected in Example 3.2.3. The results
are shown in the graph in FIG. 33. The trends were identical to
what was observed in the TSA though the absolute values of the Tm
were higher: around 62.degree. C. in DSC compared to around
59.degree. C. in the TSA.
3.2.5 Melting Temperature of 231IL0075 in a Range of Formulation
Buffers to Explore a Wide Range of Excipients Measured by Thermal
Shift Assay
[0879] A second design of experiments (DOE) was prepared to study
the influence of some combinations of excipients on the
thermodynamic stability of 23IL0075. The combination of a sugar or
a polyol (mannitol, sucrose or sorbitol), with an amino acid
(Glycine or arginine/glutamic acid mixture), and a non-ionic
detergent (Tween 80, Tween 20 or P-F68) were studied. These
combinations were tested in three buffers (10 mM Acetate pH 5.5, 10
mM Histidine pH 6.0 and 10 mM Phosphate pH 6.0) (see Table 35).
Based on the measured melting temperatures, the optimal buffer
compositions were calculated (see Table 36). Highest melting
temperatures were predicted for the histidine buffer, the lowest
melting temperatures for the phosphate buffer. For all three
buffers sucrose came out as the best excipient, the mixture of
arginine and glutamic acid as the worst. Mannitol and sorbitol,
alone or in combination with glycine were also good as excipient,
though always gave a little bit lower Tm than sucrose. Glycine
alone was not so efficient.
Example 3.3
Solubility of the Nanobodies
3.3.1 Concentration Experiments to Determine the Solubility of
23IL0064
[0880] The IL23 Nanobody 23IL0064, respectively in D-PBS buffer, in
NaPhosphate 10 mM pH 7 with 50 mM NaCl, and in L-histidine 40 mM pH
6 with 50 mM NaCl, was concentrated stepwise with a Vivaspin
concentrator (Vivascience 5,000 MWCO 0.5-mL and 5-mL
concentrators). During the concentration experiment, the retentate
was regularly gently mixed and the concentration determined by
OD280 measurements. The presence of insoluble aggregates in the
retentate was verified by checking the protein concentration before
and after centrifugation at maximum speed in a benchtop Eppendorf
centrifuge. In all three buffers a concentration of 50 mg/mL could
be reached without visual precipitation. Then the protein solution
was transferred from the 5 mL to the 0.5 mL Vivaspin.RTM.
concentrator and concentrated at a much higher centrifugal force
(15000.times.g instead of 1500.times.g). This caused rapid further
concentration, and local concentrations close to the membrane were
even much higher than the average concentration measured after
recovery of the retentate. In Table 27, the final concentrations
achieved are summarized, obtained after pipetting up and down the
retentate, and centrifugation to remove precipitate.
[0881] All samples were also analyzed by SE-HPLC. Concentrating in
D-PBS buffer resulted in an increase in % pre-peak. In the 10 mM
phosphate buffer and in the 40mM Histidine buffer, both with 50 mM
NaCl, the SE-HPLC profile remained exactly the same at 83 and 150
mg/mL compared to the starting material (FIG. 34). A generic
SE-HPLC method was used on a Phenomenex BioSep SEC S-2000 column,
with D-PBS as mobile phase at 0.2 mL/min.
[0882] A sample of the concentrated 23IL0064 protein solutions was
diluted to 55 mg/mL using the respective buffers for further use in
the PEG precipitation method.
3.3.2 Determination of the Theoretical Solubility of 23IL0064
[0883] The theoretical solubility of 23IL0064 was determined using
the PEG exclusion method in D-PBS buffer, in NaPhosphate 10 mM pH
7, NaCl 50 mM, and in L-histidine 40 mM pH 6, NaCl 50 mM. Briefly,
a concentrated Nanobody solution (55 mg/mL) in the respective
buffer was incubated for 15 minutes at room temperature in the
presence of increasing concentrations of PEG6000. After
centrifugation at 20000.times.g for 3 minutes, log values of the
[soluble protein concentration] were plotted versus PEG6000
concentration (FIG. 35). By regression analysis and extrapolation
to a zero concentration of PEG6000, the theoretical maximum protein
concentration (and thus solubility values) could be obtained for
the Nanobody in the buffers tested.
[0884] When extrapolating from the regression plots to a zero
concentration of PEG6000, a theoretical solubility value of 60
mg/mL and of 288 mg/mL was calculated for 2311.0064 in D-PBS and in
10 mM phosphate buffer/50 mM NaCl respectively. 23IL0064 showed
extremely good solubility in the histidine buffer: in the
experiment starting from 55 mg/mL only at a PEG concentration of
27% the protein started to precipitate slightly. Therefore the
experiment was repeated with the protein dissolved at 150 mg/mL.
There only at 10% PEG precipitation occurred, but then no volume
was left for OD measurements. So the solubility was actually too
high to obtain a value in this assay.
[0885] In conclusion highest solubility was obtained in 40 mM
Histidine pH 6 with 50 mM NaCl. In phosphate buffer with 50 mM
NaCl, the solubility was better than in D-PBS (with 137 mM
NaCl).
3.3.3 Determination of the Theoretical Solubility of 2311.0064 and
23IL0075
[0886] The theoretical solubility was determined for 23IL0064 and
23IL0075 using the PEG exclusion method in NaPhosphate 10 mM pH7,
NaCl 50 mM, and in L-histidine 40 mM pH 6, NaCl 50 mM (the same
buffers as used in the previous solubility study for 23IL0064
described above). The graphs (FIG. 36 and FIG. 37) representing the
protein concentration in the supernatant as a function of the % PEG
concentration were very similar as obtained in the previous
experiment. Again the apparent solubility in histidine was higher
than in the phosphate buffer, and could not be calculated due to
minimal precipitation under the protein (5 mg/mL) and PEG
concentrations (26.7%) used. The calculated solubility in phosphate
buffer pH 7 for 23IL0064 and 23IL0075 were lower than in the
previous experiment, i.e. 55 and 42 mg/mL respectively, while we
observed up to 288 mg/mL in the earlier experiment. We must stress
though that these experiments were performed pipetting extremely
low volumes of highly viscous solutions, and therefore the absolute
numbers of solubility should be confirmed with other
techniques.
Example 3.4
Stressed Stability Studies for 23IL0064 in D-PBS
[0887] An initial 37.degree. C. stressed stability study was
performed in D-PBS. The original batch was sterilized through a
0.22 .mu.m filter and 500 .mu.A was stored at 37.degree. C. in 1.5
mL-eppendorf vials for each time point (4, 8, 12, 16, 20 and 24
weeks). Additionally approximately 9.times.100 .mu.l was stored at
-20.degree. C. (reference). A first sample was already analyzed
after 3 weeks using SE-HPLC and a pre-peak of aggregates was
detected (3%) (FIG. 38). After 4 weeks at 37.degree. C., the total
peak area on SE-HPLC and on RP-HPLC was reduced to only half of the
reference sample (FIGS. 38 and 39). We therefore decided to
prematurely terminate the stability study. In some of the remaining
samples the content was still measured by OD280 after
centrifugation. The loss of material in the 4w-37.degree. C.-sample
through precipitation (all samples were centrifuged before
analysis) was confirmed in the additional 3 samples stressed for 6
weeks at 37.degree. C.: in 2 of the 3 samples half of the material
was lost (Table 29).
[0888] It can be concluded that 23IL0064 in D-PBS easily formed
aggregates which precipitate following storage for 4 weeks on at
37.degree. C. In the RP-HPLC analysis of the 37.degree. C.-stressed
sample also 12% post-peak was observed corresponding to N-terminal
pyro-glutamate formation (FIG. 41).
Example 3.5
Stressed Stability Study for 23110064 in Histidine Buffer
[0889] From the results described in previous Examples on the
solubility, the thermal shift assay, and the 37.degree. C.-stressed
stability in D-PBS for 23IL0064, it was concluded that phosphate
was not the optimal buffer for formulation of 23IL0064. In the TSA
described in Example 3.2.2, we explored some potential formulation
buffers and the highest Tm's were obtained in histidine pH 6.5,
Hepes pH 7, and Hepes pH 8. In the solubility experiment (see
Example 3.3) the solubility in a 40 mM histidine pH 6, with 50 mM
NaCl was very high. We therefore decided to test the storage
stability in histidine buffer. In Table 30 a list of tested
formulation buffers is given. The goal of this set-up was to
compare histidine pH 6.5 with histidine pH 6, investigate the
influence of some commonly used excipients, and the influence of a
higher concentration on the stability (difference between 5 mg/mL
and 22 mg/mL). One sample in Hepes pH 8 was also included, only to
be tested after 3 weeks at 37.degree. C.
[0890] For this study, 3.2 mL of the original batch was dialyzed to
the 20 mM Hepes buffer pH 8 and approximately 65 mL (approx. 246 g)
was dialyzed to the 20 mM Histidine buffer pH 6.5. The excipients
were added in concentrated solutions (2.times.), and the sample at
pH 6 was prepared by adding HCl. The samples were then concentrated
to approximately 5 mg/mL, sterilized through a 0.22 .mu.m filter
and aliquoted in 1.5 mL-eppendorfs (500 .mu.L/eppendorf) for
storage under the different conditions.
3.5.1 Stability During Freeze Thaw
[0891] One sample of each of above formulations in histidine was
subjected to 10 freeze/thaw cycles. The samples were analyzed by
RP-HPLC and OD280 content. No difference with the reference sample
(one freeze/thaw) was observed.
3.5.2 Shear Stress
[0892] Two samples of each of above formulations in histidine were
subjected to shear stress. The test was conducted in a cold room
(4-8.degree. C.) in small glass tubes with 300 .mu.l of protein
solution, stirred through a magnetic bar for 4 and 8 hours at a
medium rotation speed.
[0893] In all samples stressed with 4 and 8 hours of shearing clear
opalescence was present. This was quantified by OD280 content
analysis after centrifugation of the samples. Analysis by SE-HPLC
did not reveal any aggregates. RP-HPLC analysis after 4 hours of
shear stress showed no degradation, but after 8 hours of stress
some increase of the non-resolved pre-peak appeared, especially in
the concentrated sample. Also on SDS-PAGE generally no degradation
was detected. In Table 31a crude ranking based on the opalescence
and material loss in the content analysis is given.
3.5.3 Storage at 4.degree. C., 25.degree. C. and 37.degree. C. for
6 Weeks
[0894] The first analysis was performed after 2.5 weeks at
25.degree. C. and 37.degree. C. storage. The samples were analyzed
on RP-HPLC (see also Table 32), SE-HPLC, SDS-PAGE and OD 280/350.
Very little degradation was observed after 2.5 weeks (data not
shown). The results after 6 weeks of storage at 37.degree. C. are
discussed below (for the sample in Hepes pH 8, the 2.5 weeks
37.degree. C. results are discussed).
RP-HPLC Analysis
[0895] RP-HPLC analysis was performed mainly to detect chemical
degradation. Stress at 25.degree. C. and at 37.degree. C. typically
caused increase of the post-peak corresponding to the N-terminal
pyroglutamate. This post-peak increased less in the histidine
buffer pH 6 than in pH 6.5, and fastest in the Hepes buffer pH 8:
e.g. after 2.5 weeks at 37.degree. C. there was 11% pyroglutamate
post peak in Hepes pH 8, 7% in histidine pH 6.5, and 5% in
histidine pH 6 (data not shown).
[0896] Further a second unknown post-peak appeared. In FIG. 40 an
overview of the integration data is given. In FIG. 41 an overlay
between the chromatograms obtained for the different storage
temperatures of 23IL0064 in histidine buffer pH 6.5 at 22 mg/mL is
given.
SE-HPLC Analysis
[0897] For the samples at 5 mg/mL stressed for 6 weeks at
37.degree. C., small amounts of aggregates (between 0.5 and 1%)
were observed. The peaks were integrated. The separated pre- and
postpeaks were never higher than 1 percent.
[0898] For the sample at 22.4 mg/mL however, 3% aggregates were
detected in the sample stored at 37.degree. C.
SDS-PAGE Analysis
[0899] Analysis by SDS-PAGE showed little degradation. For the
samples stressed at 37.degree. C. for 6 weeks, a slight increase in
intensity of a degradation band at a Mw of approximately 27 kDa was
observed and some thin bands under the main band were present (FIG.
42). No difference between the different formulation buffers was
observed,
Analysis of OD280, OD350 and Sub Visible Particles
[0900] All samples in the storage stability study were further
analyzed for their Nanobody content (by OD280), for their opacity
(by OD350), and to detect subvisible particles (by PAMAS). Very
similar results were obtained between the reference and the
different temperature storage samples (data not shown).
Example 3.6
Elastic Light Scattering
[0901] The tendency for aggregate formation of 23IL0075 in the
different formulation buffers was determined using elastic light
scattering measured at an angle of 90.degree. by
temperature-induced denaturation as measured in the Jasco
Spectrofluorometer (excitation and emission wavelength 500 nm).
First we looked for the optimal protein concentration, using the 10
mM phosphate buffer pH 6.0. At concentrations of 175 .mu.g/mL
23IL0075 or lower no increase in scatter intensity was seen in the
tested temperature interval: (45-95.degree. C.). Only at 250
.mu.g/mL scatter was observed. The curve seemed to display two
transitions, which could indicate the formation of two different
types of aggregates (see FIG. 43).
[0902] The experiment was repeated for the acetate and the
histidine buffers, both at pH 6.0. The aggregation onset
temperatures in the three buffers were very similar (Table 37). The
main difference between the three buffers was the maximum scatter:
it stayed within detector range (around 435 abs) for histidine
while it went out of range in the phosphate as well as in the
acetate buffers (FIGS. 44 and 45). In histidine the second
transition was absent (FIG. 46). As the scatter is proportional to
the level of aggregates formed, this indicated that the 10 mM
histidine would be a more optimal formulation buffer than 10 mM
acetate and 10 mM phosphate pH 6.
Example 3.7
Freeze/Thaw and Shear Stress Study on 23IL0075 in a Histidine,
Acetate and Phosphate Formulation Buffer with Mannitol or a Mixture
of a Mannitol and Glycine as Excipients, and a Non-Ionic Detergent
as Surfactant
[0903] The sensitivity of 23IL0075 to freeze/thawing and to shear
or stirring has been investigated in different candidate
formulation buffers (see Table 38). The freeze/thaw stress study
consisted of 10 cycles: 100 .mu.l sample in an eppendorf tube was
frozen at -20.degree. C. until completely frozen, and thawed at
room temperature for 30 minutes followed by gentle mixing. The
shear stress test was conducted in a cold room (4-8.degree. C.) in
small glass tubes with 150 .mu.l of protein solution; the protein
solution was stirred through a magnetic bar for 4 hours at a medium
rotation speed. All samples were analyzed by RP-HPLC, SE-HPLC and
OD500, some samples also by Biacore. On RP-HPLC, no influence of
shear or freeze/thaw was detected.
[0904] On SE-HPLC, dependent on the formulation buffer, freeze/thaw
stress caused an increase in % pre-peak up to 2.5% (FIG. 47 (B)). A
mixture of mannitol and glycine protected better against
freeze/thaw stress than only mannitol as excipient (FIG. 47).
[0905] In the shear stressed samples hardly any increase in %
pre-peak on SE-HPLC was detected but up to 10 times more
opalescence (OD500) was measured than in the freeze/thaw samples.
Optical density at 500 nm (OD500) increased in the stirred samples
and correlated with the opalescence in the samples. We conclude
that in the histidine buffer with 0.05% Poloxamer or with 0.005%
Tween 80 the opalescence remained lowest (FIG. 48).
Example 3.8
Freeze/Thaw, Shear Stress and Temperature Stress (37.degree. C.)
Stability Study for 23IL0075 in a Histidine Formulation Buffer with
Different Combinations of Excipients
[0906] Based on the conclusions of Example 3.7, we further tested
freeze/thaw, shear stress and temperature stress (37.degree. C.)
stability in different histidine formulation buffers (FIG. 49 and
FIG. 50). Different excipients were tested in a formulation with 25
mg/mL of protein. An overview of the formulation buffers tested in
F/T and storage stability is presented in Table 39. In FIG. 49 the
results of OD500 measurements and SE-HPLC after freeze/thaw stress
are presented. A negligible increase of OD500 was observed. In
SE-HPLC, we saw an increase of oligomers only for the sample with
5.4% mannitol as excipient. Table 40 presents the tested buffers
for shear stress. Here no detergents were included, to mimic the
situation during the final concentration step of the DSP process.
In FIG. 50 the OD500, SE-HPLC and Biacore results obtained after 4
hours of stirring of the formulation are presented. Stirring of the
sample caused increase in OD500 absorption, but no influence on the
% soluble oligomers as measured by SE-HPLC. These samples were also
tested for albumin binding on Biacore. We conclude that the protein
was best protected against the shear stress by 10% sucrose,
followed by a mixture of mannitol and glycine as excipients. By
comparison of the values with the values of Example 3.7, we see
that the results were reproducible, and that for the shear stress
the addition of some detergent was beneficial.
[0907] The accelerated stability samples at 25 mg/mL in the
different candidate formulation buffers were analyzed by OD500,
SE-HPLC and RP-HPLC after 3 and 6 weeks storage. In SE-HPLC, the
increase of oligomers was only seen at 37.degree. C. (FIG. 51). In
RP-HPLC the post-peak corresponding to the pyroglutamate increased
from 3% to 4% after 6 weeks storage at 25.degree. C., but to on
average 9% after 6 weeks at 37.degree. C. In Table 41 the RP-HPLC
and SE-HPLC results after 3 and 6 weeks storage at 37.degree. C.
and 6 weeks at 25.degree. C. are shown. The OD500 values remained
for all buffers (except one outlier) below 0.01.
Tables
TABLE-US-00001 [0908] TABLE 1 Overview of the different formulation
buffers of RANKL008a used in stability testing. Concentration
[NaCl] Mannitol Buffer RANKL008a (mg/mL) Buffer (mM) % (w:v) 1 60
10 mM NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 50 0 2 60 10 mM
NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 100 0 3 60 10 mM
NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 0 10 4 59 10 mM Na-acetate,
pH 5.5 50 0 5 59 10 mM Na-acetate, pH 5.5 100 0 6 59 10 mM
Na-acetate, pH 5.5 0 10 7 60 20 mM L-histidine, pH 5.5 50 0 8 60 20
mM L-histidine, pH 5.5 100 0 9 60 20 mM L-histidine, pH 5.5 0 10 10
58 20 mM L-histidine, pH 6 50 0 11 58 20 mM L-histidine, pH 6 100 0
12 58 20 mM L-histidine, pH 6 0 10 13 84.3 10 mM
NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 100 0 14 70 10 mM
NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 0 5
TABLE-US-00002 TABLE 2 Relative potencies of HSA and RANKL binding
moieties of RANKL008a after 10 F/T cycles as determined by the
ELISA potency assays (inhibition and HSA binding). Relative potency
(relative to reference material) Buffer RANKL HSA Phosphate + 50 mM
NaCl, pH 7 0.904 0.767 Phosphate + 100 mM NaCl, pH 7 0.966 0.672
Phosphate + 10% Mannitol, pH 7 0.956 0.715 Acetate + 50 mM NaCl, pH
5.5 1.033 0.747 Acetate + 100 mM NaCl, pH 5.5 0.905 0.705 Acetate +
10% Mannitol, pH 5.5 0.878 0.737 Histidine + 50 mM NaCl, pH 5.5
0.724 0.723 Histidine + 100 mM NaCl, pH 5.5 0.719 0.670 Histidine +
10% Mannitol, pH 5.5 0.692 0.572 Histidine + 50 mM NaCl, pH 6 0.927
0.768 Histidine + 100 mM NaCl, pH 6 0.923 0.680 Histidine + 10%
Mannitol, pH 6 0.882 0.754
TABLE-US-00003 TABLE 3 Integration data (% of total surface area)
of the different peaks observed in the SE-HPLC chromatograms of
RANKL008a after 10 F/T cycles or stored at 37.degree. C. in
different formulation buffers at all time points tested and in
comparison with each control sample (each buffer). Histidine
Histidine Histidine Phosphate pH 7 Phosphate pH 7 Phosphate pH 7
Acetate pH 5.5 pH 5.5 pH 5.5 pH 5.5 Histidine pH 6 50 mM NaCl 100
mM NaCl 10% Mannitol Acetate pH 5.5 Acetate pH 5.5 10% Mannitol 50
mM 100 mM 10% Mannitol Histidine pH 6 Histidine pH 6 10% Mannitol
SE-HPLC Sample 60 mg/ml 60 mg/ml 60 mg/ml 50 mM NaCl 100 mM NaCl 59
mg/ml NaCl NaCl 60 mg/ml 50 mM NaCl 100 mM NaCl 58 mg/ml % control
0 0 0 0 0 0 0 0 0 0 0 0 Prepeak 10 F/T 0 0 0 0 0 0 0 0 0 0 0 0
cycles 2 w 37.degree. C. 5.6 6.9 1.3 4.6 6.3 2.3 5.5 7.5 0.54 6.3
7.7 0.63 3 w 37.degree. C. 4.4 6.2 0.65 3.9 5.9 0.18 5.6 7.9 0.34
7.0 8.6 0.39 5 w 37.degree. C. 13.7 15.8 3.9 11.5 14.2 1.22 14.0
17.1 1.5 16.2 17.4 2.0 10 w 37.degree. C. 23.8 25.3 11.1 21.0 23.9
3.4 27.2 27.8 5.4 26.8 27.0 7.3 % control 100 100 100 100 100 100
100 100 100* 100 100 100* Main peak 10 F/T 100 100 100 100 100 100
100 100 100 100 100 100 cycles 2 w 37.degree. C. 93.5 92.2 97.9
94.8 93.1 98.8 94.0 92.1 98.8 93.1 91.5 96.7 3 w 37.degree. C. 93.7
92.0 95.2 95.0 92.8 96.9 93.4 91.5 98.6 91.3 90.2 98.8 5 w
37.degree. C. 81.14 78.87 91.52 87.38 84.63 97.87 84.85 81.73 97.49
82.22 81.19 96.76 10 w 37.degree. C. 69.2 68.0 80.5 77.5 74.7 95.1
71.3 73.5 93.1 71.3 71.2 91.0 % control 0 0 0 0 0 0 0 0 0 0 0 0
Postpeak 1 10 F/T 0 0 0 0 0 0 0 0 0 0 0 0 cycles 2 w 37.degree. C.
0 0 0 0 0 0 0 0 0 0 0 0 3 w 37.degree. C. 0 0 0 0 0 0 0 0 0 0 0 0 5
w 37.degree. C. 3.16 3.36 3.12 0 0 0 0 0 0 0 0 0 10 w 37.degree. C.
3.7 3.5 5.0 0 0 0 0 0 0 0 0 0 % control 0 0 0 0 0 0 0 0 0 0 0 0
Postpeak 2 10 F/T 0 0 0 0 0 0 0 0 0 0 0 0 cycles 2 w 37.degree. C.
0.23 0.27 0.19 0.23 0.26 0.19 0.19 0.17 0.19 0.20 0.23 0.18 3 w
37.degree. C. 0.57 0.58 0.31 0.49 0.53 0.27 0.48 0.55 0.27 0.54 0.5
0.27 5 w 37.degree. C. 0.41 0.47 0.27 0.37 0.39 0.25 0.45 0.29 0.23
0.52 0.42 0.37 10 w 37.degree. C. 0.5 0.5 0.3 0.4 0.4 0.2 0.4 0.5
0.2 0.4 0.4 0.3 % control 0 0 0 0 0 0 0 0 0 0 0 0 Postpeak 3 10 F/T
0 0 0 0 0 0 0 0 0 0 0 0 cycles 2 w 37.degree. C. 0.62 0.64 0.60
0.37 0.41 0.46 0.31 0.26 0.37 0.40 0.58 0.53 3 w 37.degree. C. 1.15
1.25 1.07 0.52 0.64 0.61 0.49 0.55 0.57 1.12 0.71 0.56 5 w
37.degree. C. 1.59 1.50 1.49 0.75 0.78 0.66 0.70 0.88 0.78 1.06
0.99 0.87 10 w 37.degree. C. 2.7 2.6 3.1 1.1 1.0 1.3 1.1 1.3 1.3
1.5 1.4 1.5
TABLE-US-00004 TABLE 4 Integration data (% of total surface area)
of the different peaks observed in the RP-HPLC chromatograms of
RANKL008a after 10 F/T cycles or stored at 37.degree. C. in
different formulation buffers at all time points tested and in
comparison with each control sample (each buffer). Histidine
Histidine Phosphate pH 7 Phosphate pH 7 Phosphate pH 7 Acetate
Acetate Acetate pH 5.5 Histidine Histidine pH 5.5 Histidine pH 6
10% 50 mM NaCl 100 mM NaCl 10% Mannitol pH 5.5 pH 5.5 10% Mannitol
pH 5.5 pH 5.5 10% Mannitol pH 6 Histidine pH 6 Mannitol RP-HPLC
Sample 60 mg/ml 60 mg/ml 60 mg/ml 50 mM NaCl 100 mM NaCl 59 mg/ml
50 mM NaCl 100 mM NaCl 60 mg/ml 50 mM NaCl 100 mM NaCl 58 mg/ml %
control 0.25 0.14 0 0 0 ND 0 0 0 0 0 0 Prepeak 1 10 F/T 0.20 0.08 0
0 0 0 0 0 0 0 0 0 cycles 2 w 37.degree. C. 0 0 0 0 0 0 0 0 0 0 0 0
3 w 37.degree. C. 0 0 0 0 0 0 0 0 0 0 0 0 5 w 37.degree. C. 0 0 0 0
0 0 0 0 0 0 0 0 10 w 0 0 0 0 0 0 0 0 0 0 0 0 37.degree. C. %
Control 0 0 0 0 0 ND 0 0 0 0 0 0 Prepeak 2 10 F/T 0 0 0 0 0 0 0 0 0
0 0 0 cycles 2 w 37.degree. C. 1.10 1.00 1.10 0.78 0.82 0.78 0.82
0.72 0.76 0.76 0.79 0.87 3 w 37.degree. C. 1.6 1.4 1.8 0.7 0.8 0.9
0.7 0.9 0.9 0.7 0.9 0.9 5 w 37.degree. C. 2.1 2.0 2.3 1.0 0.9 1.1
1.1 1.2 1.2 1.2 1.1 1.5 10 w 3.4 2.9 3.7 1.5 1.5 1.9 1.6 1.7 2.0
2.0 1.9 2.1 37.degree. C. % Control 96.6 96.7 96.7 96.8 97.0 ND
96.5 96.6 96.4 96.7 96.8 96.8 Main peak 10 F/T 96.7 96.6 95.9 97.0
96.7 96.6 97.0 95.8 96.5 96.4 96.8 96.8 cycles 2 w 37.degree. C.
93.7 94.0 93.5 95.8 96.0 95.6 96.0 95.7 95.9 95.6 95.7 95.5 3 w
37.degree. C. 92.3 92.7 91.9 95.7 95.5 95.4 95.6 95.4 95.3 95.6
95.5 95.5 5 w 37.degree. C. 90.2 90.3 89.1 94.8 95.1 94.7 94.7 94.6
94.8 94.4 94.7 94.0 10 w 84.1 85.4 83.4 93.5 93.3 92.7 93.3 93.3
92.4 93.1 92.3 92.0 37.degree. C. % Control 3.2 3.1 3.3 3.2 2.9 ND
3.5 3.0 3.6 3.3 3.2 3.2 Pyroglut. 10 F/T 3.1 3.3 4.0 3.3 3.2 3.4
3.0 3.3 3.5 3.6 3.2 3.2 cycles 2 w 37.degree. C. 5.1 4.7 5.4 3.4
3.2 3.6 3.2 3.5 3.4 3.6 3.5 3.6 3 w 37.degree. C. 6.1 6.0 6.3 3.6
3.7 3.7 3.7 3.7 3.7 3.6 3.6 3.6 5 w 37.degree. C. 7.7 7.7 8.6 4.1
3.9 4.2 4.2 4.3 4.1 4.4 4.1 4.4 10 w 12.5 11.7 12.9 5.0 5.2 5.5 5.2
5.1 5.5 4.8 5.8 5.9 37.degree. C.
TABLE-US-00005 TABLE 5 Integration data (% of total surface area)
of the different peaks observed in the IEX-HPLC chromatograms of
RANKL008a stored for 10 weeks at 37.degree. C. in different
formulation buffers. % Main % Post % Post Buffer peak peak 1 peak 2
Phosphate + 50 mM NaCl, pH 7 69.6 4.4 26.0 Phosphate + 100 mM NaCl,
pH 7 67.0 4.5 28.1 Phosphate + 10% Mannitol, pH 7 84.6 3.2 12.1
Acetate + 50 mM NaCl, pH 5.5 72.8 2.7 24.4 Acetate + 100 mM NaCl,
pH 5.5 69.6 2.8 27.6 Acetate + 10% Mannitol, pH 5.5 95.4 0 4.6
Histidine + 50 mM NaCl, pH 5.5 66.0 2.8 31.2 Histidine + 100 mM
NaCl, pH 5.5 68.1 2.8 29.0 Histidine + 10% Mannitol, pH 5.5 92.8 0
7.2 Histidine + 50 mM NaCl, pH 6 67.4 2.6 30.1 Histidine + 100 mM
NaCl, pH 6 67.0 2.6 30.4 Histidine + 10% Mannitol, pH 6 88.8 2.6
9.0
TABLE-US-00006 TABLE 6 Relative potencies of the HSA and RANKL
binding moieties of RANK1008a after 10 weeks at 37.degree. C. as
measured by Biacore analysis. Relative potency Buffer RANKL HSA
Phosphate + 50 mM NaCl, pH 7 81.0 57.4 Phosphate + 100 mM NaCl, pH
7 78.6 56.6 Phosphate + 10% Mannitol, pH 7 76.3 66.8 Acetate + 50
mM NaCl, pH 5.5 80.1 63.0 Acetate + 100 mM NaCl, pH 5.5 78.0 59.0
Acetate + 10% Mannitol, pH 5.5 80.9 79.4 Histidine + 50 mM NaCl, pH
5.5 80.2 59.7 Histidine + 100 mM NaCl, pH 5.5 73.1 55.0 Histidine +
10% Mannitol, pH 5.5 75.2 73.6 Histidine + 50 mM NaCl, pH 6 79.1
59.3 Histidine + 100 mM NaCl, pH 6 78.3 57.5 Histidine + 10%
Mannitol, pH 6 87.4 83.4
TABLE-US-00007 TABLE 7 Visual inspection and content determination
of RANKL008a after dilution to 0.28 mg/mL in different diluents and
passage/storage in syringes (refer to FIG. 9). content (mg/mL) (95%
Sample* visual inspection confidence interval 0028 SALINE TUB small
precipitates 0.265 (0.261-0.270) 0028 SALINE SYR S25/0 small
precipitates 0.263 (0.261-0.265) 0028 SALINE SYR S25/24 small
precipitates 0.259 (0.256-0.262) 0028 PLACEBO - TW small
precipitates 0.272 (0.271-0.273) TUB 0028 PLACEBO - TW small
precipitates 0.268 (0.267-0.269) SYR S25/0 0028 PLACEBO - TW small
precipitates 0.268 (0.259-0.276) SYR S25/24 0028 PLACEBO + TW TUB
clear 0.281 (0.281-0.281) 0028 PLACEBO + TW slightly turbid 0.280
(0.278-0.282) SYR S25/0 0028 PLACEBO + TW slightly turbid 0.279
(0.277-0.281) SYR S25/24 *S25/0: storage at 25.degree. C. for 0
minute S25/24: storage at 25.degree. C. for 24 h -TW: buffer minus
Tween 80 +TW: buffer + Tween 80 PLACEBO refers to the following
buffer: 10 mM Na2HPO4 pH 7.0 + 115 mM NaCl TUB: sample stored in a
polystyrene tube
TABLE-US-00008 TABLE 8 Visual inspection, content (confidence
interval) and turbidity of RANKL008a diluted to 0.28 mg/mL before
(TUB) and after passage through syringes with different needle size
as described in Example 1.8. content (mg/mL) (95% OD OD visual
confidence 320/278 350/278 Sample* inspection interval) ratio ratio
0028 PLACEBO + clear 0.288 (0.275-0.301) 0.0010 0.0019 TW TUB 0028
PLACEBO + clear 0.285 (0.284-0.286) 0.0003 0.0000 TW 18G/18G 0028
PLACEBO + clear 0.288 (0.271-0.307) 0.0000 0.0000 TW 18G/23G 0028
PLACEBO + clear 0.285 (0.279-0.290) 0.0000 0.0002 TW 18G/27G 0028
PLACEBO + clear 0.286 (0.285-0.287) 0.0005 0.0002 TW 18G/30G 0028
PLACEBO + clear 0.287 (0.285-0.289) 0.0005 0.0007 TW 23G/23G 0028
PLACEBO + clear 0.285 (0.284-0.286) 0.0001 0.0005 TW 27G/27G 0028
PLACEBO + clear 0.287 (0.280-0.294) 0.0007 0.0019 TW 30G/30G *+TW:
buffer + Tween 80 PLACEBO refers to the following buffer: 10 mM
Na2HPO4 pH 7.0 + 115 mM NaCl TUB: sample stored in a polystyrene
tube 18G/18G: sample drawn up with a 18G needle and expelled
through a 18G needle 18G/27G: drawn up with a 18G needle and
expelled through a 27G needle All other coding is similar to the
two examples given above
TABLE-US-00009 TABLE 9 Visual inspection, content (with 95%
confidence interval) and turbidity of RANKL008a before (TUB) and
after passage through syringes with different needle size as
described in Example 1.7 at a concentration of 0.28 mg/mL or about
65 mg/mL. content (mg/mL) (95% OD OD visual confidence 320/278
350/278 Sample* inspection interval) ratio ratio 0028 PLACEBO +
clear 0.284 (0.283-0.285) 0.0014 0.0010 TW TUB 0028 PLACEBO + clear
0.284 (0.283-0.285) 0.0031 0.0021 TW 27G/27G (3x) 0028 PLACEBO +
clear 0.282 (0.280-0.284) 0.0024 0.0010 TW 29G/29G B 0028 PLACEBO +
clear 0.283 (0.282-0.284) 0.0041 0.0033 TW 29G/29G T 6500 PLACEBO +
clear 63.5 (62.4-64.6) 0.0019 0.0006 TW TUB 6500 PLACEBO + clear
62.9 (62.7-63.1) 0.0015 0.0008 TW 27G/27G (3x) *+TW: buffer + Tween
80 PLACEBO refers to the following buffer: 10 mM Na2HPO4 pH 7.0 +
115 mM NaCl TUB: sample stored in a polystyrene tube 27G/27G:
sample drawn up with a 27G needle and expelled through a 27G needle
29G/29G: drawn up with a 29G needle and expelled through a 29G
needle T: Terumo needle, B Becton Dickinson needle 0028 refers to
concentration at 0.28 mg/mL, 6500 to 65 mg/mL
TABLE-US-00010 TABLE 10 Protein sequences of Nanobodies used in
Example 2 IL6R304, SEQ ID NO: 1
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNA
KNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSL
RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSRSSQGTLVTVSS IL6R305, SEQ ID NO: 2
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNA
KNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSL
RLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAV
YYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSLRLSCAASGFTFSSFGMSW
VRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTAVYYCTIGGSLSRSSQGTL
VTVSS IL6R306, SEQ ID NO: 3
EVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPGKGRELVAGIISGGSTSYADSVKGRFTISRDNA
KNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGNSL
RLSCAASGFTFSSFGMSWVRQAPGKGLEWVSSISGSGSDTLYADSVKGRFTISRDNAKTTLYLQMNSLRPEDTA
VYYCTIGGSLSRSSQGTLVTVSSGGGGSGGGSEVQLVESGGGLVQPGGSLRLSCAASGSVFKINVMAWYRQAPG
KGRELVAGIISGGSTSYADSVKGRFTISRDNAKNTLYLQMNSLRPEDTAVYYCAFITTESDYDLGRRYWGQGTL
VTVSS
TABLE-US-00011 TABLE 11 Overview of the IL6R batches used in the
formulation and stability studies described in Example 2. Batch no.
Nanobody Buffer Concentration P#051108nr1 IL6R304 PBS 4.58 mg/mL
P#051108nr2 IL6R305 PBS 3.46 mg/mL P#051108nr2 IL6R306 PBS 5.65
mg/mL B5#030309nr1.5-9 IL6R304 25 mM hepes, pH 7.5, 3.79 mg/mL 100
mM NaCl B5#060509nr1 IL6R304 25 mM hepes, pH 7.5, 4.6 mg/mL 100 mM
NaCl
TABLE-US-00012 TABLE 12 Reagents used in the formulation and
stability study described in Example 2 Reagent Provider Cat No.
ACN, HPLC grade Biosolve Cat. No. 012007 TFA Biosolve Cat. No.
20234131 Isopropanol, HPLC grade Biosolve Cat. No. 162606 MilliQ
grade water D-PBS Gibco Cat. No. 14190-094 NaCl Merck Cat. No.
1.06404.1000 Gel filtration standard Bio-Rad Cat. No. 151-1901 HSA
Sigma Cat. No. A3782 L-histidine Fluka Cat. No. 53319 D-Mannitol
Fluka Cat. No. 17311 Sucrose Fluka Cat. No. 18219 Glycine Fluka
Cat. No. 50058 Tween-80 Merck Cat. No. K351 65661 609
L-histidine-HCl monohydrate Sigma Cat. No. 53369 Succinic acid
disodium Fluka Cat. No. 14158 hexahydrate Trizma base Sigma Cat.
No. T6066-5 Sorbitol Fluka Cat. No. 85529 Xylitol Sigma Cat. No.
X3375-100g Ribitol Fluka Cat. No. 02240 L-Arginine Fluka Cat. No.
11009 MES Sigma Cat. No. M3671 Sodium dihydrogenphosphate Merck
Cat. No. 1.06345.1000 Disodium hydrogenphosphate Merck Cat. No.
1.06576.1000
TABLE-US-00013 TABLE 13 Summary of the results from the TSA assay.
The Tm values obtained in the different buffer are coded from white
to dark grey, i.e. from higher to lower Tm values. pH Histidine
pH6.5 Hepes pH8 Phos. Succinate Histidine pH5.5 Tris pH7 Buffer
Hepes pH7 Phos. pH6.7 pH7.7 pH6.2 Succinate pH5.2 PBS NaCl 0 100
200 300 400 500 Mannitol 7.5% 5% 2.5% 0 Sucrose 10% 5% Glycine 200
mM 100 mM
TABLE-US-00014 TABLE 14 Overview of the Tm values obtained by DSC
using IL6R304. Tm Tm (.degree. C.) in (.degree. C.) in Tm (.degree.
C.) in Tm (.degree. C.) in 0 mM 25 mM 100 mM 500 mM Buffer NaCl
NaCl NaCl NaCl 25 mM citrate pH 3.5 50.21 / / / 25 mM acetate pH
5.5 61.30 61.21 60.19 58.59 25 mM MES pH 6.0 62.52 61.83 60.60
58.66 25 mM hepes pH 7.0 62.48 62.27 61.13 59.24 25 mM phosphate pH
7.0 60.70 / / / 25 mM Tris pH 7.5 61.82 61.82 60.91 59.43
TABLE-US-00015 TABLE 15 Overview of the Tm results obtained by DSC
and TSA using IL6R304 formulated in different buffers. DSC TSA Tm
(.degree. C.) Tm (.degree. C.) Tm (.degree. C.) Tm (.degree. C.) 15
mM L- 15 mM in 15 mM 15 mM histidine, phosphate, L-histidine,
phosphate, Excipient(s) pH 6.5 pH 6.5 pH 6.5 pH 6.5 / ND ND
61.09*/61.11** 60.48/60.48 5% mannitol 64.74 63.81 61.91/61.87
61.34/61.31 10% sucrose 65.40 64.35 62.78/63.20 62.34/62.78 10%
trehalose 65.28 64.51 62.78/63.59 63.17/63.61 2.5% 64.37 63.83
61.52/61.91 61.53/61.30 mannitol + 2.5% sucrose 2.5% sorbitol +
64.65 63.85 62.34/62.34 62.77/62.77 2.5% trehalose 2.5% sorbitol +
64.83 64.33 62.33/62.35 63.19/62.96 2.5% trehalose + 1.5 mM Glycine
10% Run 63.77 ND ND trehalose + failure 0.01% Tween- 80
*measurement 1, **measurement 2
TABLE-US-00016 TABLE 16 Visual appearance, UV spectroscopy and
PAMAS data demonstrating a higher solubility for IL6R304 in the
presence of Tween 80. IL6R304 (2 mg/mL) stored for 4 days at
5.degree. C. PBS + 0.1% Tween PBS + 0.2% Tween PBS 80 80 Appearance
Turbid, Clear, colorless Clear, colorless opaque A320/A280 0.014
0.012 0.009 Sub-visible particle counts/100 .mu.l >1 .mu.m
157.557 42.243 52.157 >2 .mu.m 69.471 19.514 21.429 >3 .mu.m
43.371 12.043 12.743 >4 .mu.m 29.757 8.329 8.600 >5 .mu.m
18.300 4.971 4.800 >6 .mu.m 11.814 3.114 3.143 >7 .mu.m 8.300
2.300 2.186 >8 .mu.m 5.900 1.671 1.500 >9 .mu.m 4.543 1.214
1.086 >10 .mu.m 3.400 971 800 >15 .mu.m 800 271 300 >25
.mu.m 200 100 86 >50 .mu.m 114 29 14 >100 .mu.m 86 0 14
>150 .mu.m 71 0 14 >200 .mu.m 71 0 14
TABLE-US-00017 TABLE 17 Overview of the different formulation
buffers used in initial stability testing of IL6R304, IL6R305 and
IL6R306. Condition Buffer [NaCl] Mannitol 1 PBS 0 mM 0% 2 PBS 0 mM
5% 3 10 mM NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 100 mM 0% 4 10
mM NaH.sub.2PO.sub.4.cndot.2H.sub.2O, pH 7 100 mM 5% 5 10 mM
Na-acetate, pH 5.5 100 mM 0% 6 10 mM Na-acetate, pH 5.5 100 mM 5% 7
20 mM L-histidine, pH 6 100 mM 0% 8 20 mM L-histidine, pH 6 100 mM
5%
TABLE-US-00018 TABLE 18 Overview of the different formulation
buffers used in stability testing of IL6R304. Concen- % tration
Tween % % mM Buffer IL6R304 Buffer 80 Mannitol Sucrose Glycine 1 10
mg/mL 20 mM L- / / / / histidine 2 10 mg/mL 20 mM L- 0.01 / / /
histidine 3 10 mg/mL 20 mM L- 0.05 / / / histidine 4 10 mg/mL 20 mM
L- 0.05 5 / / histidine 5 10 mg/mL 20 mM L- 0.05 5 / 200 histidine
6 10 mg/mL 20 mM L- 0.05 2.5 / 100 histidine 7 10 mg/mL 20 mM L-
0.05 / 10 / histidine 8 10 mg/mL 20 mM L- 0.05 / / 200 histidine 9
10 mg/mL 20 mM L- 0.05 / 5 100 histidine 10 10 mg/mL 20 mM L- 0.05
2.5 5 / histidine 11 10 mg/mL 20 mM L- / 2.5 5 100 histidine 12 10
mg/mL 20 mM L- 0.05 2.5 5 100 histidine
TABLE-US-00019 TABLE 19 Methods used for assessing the stability of
IL6R304 at different time points (represented as x weeks or w)
after storage at 5.degree. C. and 37.degree. C. Ref. Stress
condition Method Purpose material 5.degree. C. 37.degree. C. A280
Content 0 w 1, 2 and 5 w 1, 2, 3 and 5 w Appearance Precipitation 0
w 1, 2 and 5 w 1, 2, 3 and 5 w RP-HPLC Purity/ 0 w 1, 2 and 5 w 1,
2, 3 and 5 w variants SE-HPLC Purity/ 0 w 1, 2 and 5 w 1, 2, 3 and
5 w aggregation/ hydrolysis Biacore Potency 0 w 5 w 5 w (HSA
binding) Osmolality Characteristic 0 w / /
TABLE-US-00020 TABLE 20 Overview of the SE-HPLC integration results
after storage for 6 months at 37.degree. C. Buffer % pre peak 1 %
pre peak 2 % main peak % post peak Ref 0.52 0.17 99.3 0 Buffer 1 ND
ND ND ND Buffer 2 20.4 2.1 73.4 4.1 Buffer 3 ND ND ND ND Buffer 4
18.1 1.7 76.0 4.2 Buffer 5 22.2 2.0 71.4 4.4 Buffer 6 21.4 1.7 72.7
4.2 Buffer 7 15.1 0 80.5 4.4 Buffer 8 21.1 2.4 72.0 4.5 Buffer 9
16.7 2.7 76.3 4.3 Buffer 10 15.8 1.9 77.9 4.4 Buffer 11 17.5 2.0
76.4 4.2 Buffer 12 16.8 3.3 75.7 4.2
TABLE-US-00021 TABLE 21 Overview of the different formulation
buffers tested in the stability study. Nr. Conc. Buffer Mannitol
Sucrose Trehalose Glycine Tween-80 1 10 mg/mL 15 mM L-histidine,
pH6.5 5% 0.01% 2 10 mg/mL 15 mM L-histidine, pH6.5 10% 0.01% 3 10
mg/mL 15 mM L-histidine, pH6.5 10% 0.01% 4 10 mg/mL 15 mM
L-histidine, pH6.5 7.5% 0.35% 0.01% 5 10 mg/mL 15 mM L-histidine,
pH6.5 2.5% 5% 0.01% 6 10 mg/mL 15 mM phosphate, pH6.5 5% 0.01% 7 10
mg/mL 15 mM phosphate, pH6.5 10% 0.01% 8 10 mg/mL 15 mM phosphate,
pH6.5 10% 0.01% 9 10 mg/mL 15 mM phosphate, pH6.5 7.5% 0.35% 0.01%
10 10 mg/mL 15 mM phosphate, pH6.5 2.5% 5% 0.01%
TABLE-US-00022 TABLE 22 cIEF integration data of IL6R304 stored for
8 weeks at 37.degree. C. in the different buffers % pre peak % post
peak Buffer (acidic variants) % main peak (basic variants) 1 5.5
81.3 13.0 2 5.0 81.5 13.5 3 6.1 79.7 14.2 4 5.7 81.2 13.2 5 5.1
81.2 13.7 6 9.0 71.6 19.3 7 9.9 70.5 19.6 8 8.3 71.8 19.9 9 11.7
68.5 19.8 10 8.7 70.5 20.2
TABLE-US-00023 TABLE 23 Relative potency of IL6R304 after 8 weeks
at +37.degree. C. compared to B5#030309nr2.3-5. Buffer HSA IL-6R 1
1.080 (0.954-1.223) 1.153 (0.957-1.389) 2 0.975 (0.887-1.072) 0.980
(0.760-1.263) 3 1.038 (0.952-1.132) 1.117 (0.910-1.372) 4 1.182
(1.074-1.300) 1.061 (0.908-1.240) 5 1.080 (1.004-1.161) 1.082
(0.925-1.266)
TABLE-US-00024 TABLE 24 Summary of the Biacore results for HSA
binding of the stability samples stored for 8 weeks at 37.degree.
C., expressed as % activity compared to the equivalent sample
stored at -70.degree. C. Buffer % activity compared to reference 1
97.5 2 93.2 3 92.5 4 83.9 5 101.9 6 92.2 7 89.4 8 99.0 9 84.3 10
89.6
TABLE-US-00025 TABLE 25 Appearance of IL6R304 after 0, 2, 4 and 24
hours of stirring at 2-8.degree. C. Buffer 0 hrs 2 hrs 4 hrs 24 hrs
1 clear clear clear clear/slightly opalescent 2 clear clear clear
clear/slightly opalescent 3 clear clear slightly opalescent
slightly opalescent 4 clear clear clear clear/slightly opalescent 5
clear clear slightly opalescent slightly opalescent 6 clear clear
slightly opalescent opalescent 7 clear clear slightly opalescent
opalescent 8 clear clear slightly opalescent highly opalescent 9
clear clear clear opalescent 10 clear clear opalescent
opalescent
TABLE-US-00026 TABLE 26 Reagents used in the formulation and
stability study described in Examples 3. Reagent Provider Cat No.
ACN, HPLC grade Biosolve Cat. No. 012007 TFA Biosolve Cat. No.
20234131 N-propanol, HPLC grade Sigma-Aldrich Cat. No. 34871 MilliQ
grade water D-PBS Invitrogen Cat. No. 14190 NaCl Merck Cat. No.
1.06404.1000 Gel filtration standard Bio-Rad Cat. No. 151-1901 HSA
Sigma Cat. No. A3782 L-histidine Fluka Cat. No. 53319 D-Mannitol
Fluka Cat. No. 17311 Sucrose Fluka Cat. No. 18219 Glycine Fluka
Cat. No. 50058 Tween 80 Merck Cat. No. K351 65661 609
TABLE-US-00027 TABLE 27 Final concentrations obtained after
concentration of 23IL0064 using Vivaspin filters. The filtration
was stopped at the moment the final volume became limited (100 to
200 .mu.L) and protein loss occurred. All samples were analyzed by
SE-HPLC: the percent pre-peak (% of total surface area) represent
aggregates in the sample. start conditions after ULTRAFILTRATION %
pre-peak % pre-peak buffer condition concentration in SE-HPLC
concentration in SE-HPLC % recovery D-PBS 3.8 mg/mL 2.80% 110 mg/mL
6.30% 42%.sup.(1) 50 mM NaCl, 3.4 mg/mL 2.70% 83 mg/mL 2.80%
66%.sup.(1) 10 mM Phosphate pH 7 50 mM NaCl, 3.4 mg/mL 2.70% 150
mg/mL 2.70% 59%.sup.(1) 40 mM Histidine pH 6 .sup.(1)Low recoveries
can be due to local concentration effects obtained during the
dead-end ultrafiltration set-up.
TABLE-US-00028 TABLE 28 Buffers tested in a thermal shift assay for
23IL0064. Con- cen- tration Buffer pKa pH (mM) mM NaCl % mannitol
Succinate 5.64 (pK2) 5.2 20 0 0-2.5-5-7.5 6.2 50-150-300-500 0
Histidine 6.04 (pK2) 5.5 20 0 0-2.5-5-7.5 6.5 50-150-300-500 0
Phosphate 7.20 (pK2) 6.7 20 0 0-2.5-5-7.5 7.7 50-150-300-500 0
hepes 7.48 7.0 20 0 0-2.5-5-7.5 8.0 50-150-300-500 0
TABLE-US-00029 TABLE 29 Concentrations measured by Nanodrop
(average of 2 measurements) in the samples stressed at 37.degree.
C. The concentration was determined after a short high speed spin
(1 min at 15000xg) P23IL0064 Samples (4 mg/mL in D-PBS)
Concentration (mg/mL) Reference (-20.degree. C.) 4.05 3 weeks
37.degree. C. (label 24 w) 3.99 4 weeks 37.degree. C. (label 4 w)
2.0 6 weeks 37.degree. C. (label 8 w) 2.30 6 weeks 37.degree. C.
(label 12 w) 4.11 6 weeks 37.degree. C. (label 16 w) 2.33
TABLE-US-00030 TABLE 30 Buffers tested in stressed stability for
23IL0064. Excipient/ Time point Storage Buffer pH Concentration*
surfactant analyzed Temp. Hepes 20 mM 8 5 mg/mL 2.5 w 37.degree. C.
His 20 mM 6.5 5 mg/mL 2.5 w/6 w 4-25-37.degree. C. His 20 mM 6.5 22
mg/mL** 2.5 w/6 w 4-25-37.degree. C. His 20 mM 6 5 mg/mL 2.5 w/6 w
4-25-37.degree. C. His 20 mM 6.5 5 mg/mL 0.02% Tween 80 2.5 w/6 w
4-25-37.degree. C. His 20 mM 6.5 5 mg/mL 8% mannitol 2.5 w/6 w
4-25-37.degree. C. His 20 mM 6.5 5 mg/mL 8% sucrose 2.5 w/6 w
4-25-37.degree. C. His 20 mM 6.5 5 mg/mL 1.5% glycine 2.5 w/6 w
4-25-37.degree. C. *The exact concentrations used in the study
ranged between 4.9 and 5.1 mg/mL **Labeled as `HIGH CONC` in
figures, actual conc. was 22.36 mg/mL.
TABLE-US-00031 TABLE 31 Crude ranking of the degree of opalescence
and material loss induced by shear stress for 23IL0064 in different
formulation buffers. 5 mg/mL 20 mM Histidine pH 6.5 + 8% sucrose
Lower 20 mM Histidine pH 6.5 + 8% mannitol | 20 mM Histidine pH 6.0
| 20 mM Histidine pH 6.5 + 1.5% glycine | 20 mM Histidine pH 6.5 +
0.02% Tween 80 | 20 mM Histidine pH 6.5 .dwnarw. 20 mM Histidine pH
6.5 CONC (22.36 mg/ml) Higher opalescence
TABLE-US-00032 TABLE 32 Integration data of the RP-HPLC analysis of
the stability samples of 23IL0064 in different buffer conditions
(comparison of 6 weeks 37.degree. C., 6 weeks 25.degree. C., 6
weeks 4.degree. C. and -80.degree. C. Ref). Stress pre 1 pre 2 main
peak post 1 post 2 Total peak p23IL0064 in condition 22.4 23.3 23.9
27.5 29.0 area (%)* 20 mM -80.degree. C. Ref 68 195 1671 76 20 2029
Histidine pH % 3% 10% 82% 4% 1% 100% 6.5 6 weeks 4.degree. C. 77
201 1791 85 29 2182 % 4% 9% 82% 4% 1% 108% 6 weeks 25.degree. C. 77
196 1696 125 33 2127 % 4% 9% 80% 6% 2% 105% 6 weeks 37.degree. C.
130 186 1499 274 57 2146 % 6% 9% 70% 13% 3% 106% 20 mM -80.degree.
C. Ref 68 199 1807 66 20 2159 Histidine pH % 3% 9% 84% 3% 1% 100%
6.0 6 weeks 4.degree. C. 69 195 1751 71 17 2104 % 3% 9% 83% 3% 1%
97% 6 weeks 25.degree. C. 67 206 1696 90 22 2080 % 3% 10% 82% 4% 1%
96% 6 weeks 37.degree. C. 117 200 1573 152 37 2079 % 6% 10% 76% 7%
2% 96% 20 mM -80.degree. C. Ref 66 175 1564 49 14 1867 Histidine pH
% 4% 9% 84% 3% 1% 100% 6.5 + 6 weeks 4.degree. C. 74 196 1749 85 29
2133 0.02% % 3% 9% 82% 4% 1% 114% Tween80 6 weeks 25.degree. C. 86
196 1556 117 37 1992 % 4% 10% 78% 6% 2% 107% 6 weeks 37.degree. C.
116 195 1258 245 66 1881 % 6% 10% 67% 13% 4% 101% 20 mM -80.degree.
C. Ref 70 201 1770 66 19 2128 Histidine pH % 3% 9% 83% 3% 1% 100%
6.5 + 6 weeks 4.degree. C. 68 193 1739 80 14 2093 8% mannitol % 3%
9% 83% 4% 1% 98% 6 weeks 25.degree. C. 82 192 1665 123 32 2094 % 4%
9% 80% 6% 2% 98% 6 weeks 37.degree. C. 95 173 1438 256 35 1998 % 5%
9% 72% 13% 2% 94% 20 mM -80.degree. C. Ref 56 193 1685 70 16 2019
Histidine pH % 3% 10% 83% 3% 1% 100% 6.5 + 6 weeks 4.degree. C. 61
198 1691 62 17 2029 8% sucrose % 3% 10% 83% 3% 1% 100% 6 weeks
25.degree. C. 80 202 1591 123 29 2026 % 4% 10% 79% 6% 1% 100% 6
weeks 37.degree. C. 139 246 1292 316 73 2065 % 7% 12% 63% 15% 4%
102% 20 mM -80.degree. C. Ref 53 167 1413 51 15 1698 Histidine pH %
3% 10% 83% 3% 1% 100% 6.5 + 6 weeks 4.degree. C. 68 197 1669 67 18
2019 1.5% glycine % 3% 10% 83% 3% 1% 119% 6 weeks 25.degree. C. 74
189 1596 124 31 2014 % 4% 9% 79% 6% 2% 119% 6 weeks 37.degree. C.
95 185 1359 278 52 1968 % 5% 9% 69% 14% 3% 116% 20 mM -80.degree.
C. Ref 72 233 1984 81 15 2385 Histidine pH % 3% 10% 83% 3% 1% 100%
6.5 CONC 6 weeks 4.degree. C. 90 218 1880 88 21 2297 % 4% 9% 82% 4%
1% 96% 6 weeks 25.degree. C. 96 235 1848 143 33 2355 % 4% 10% 78%
6% 1% 99% 6 weeks 37.degree. C. 126 261 1725 332 62 2505 % 5% 10%
69% 13% 2% 105% (%)*: recovery calculated by using the total area
compared to the -80.degree. C. Ref of the same condition.
TABLE-US-00033 TABLE 33 Melting temperatures for 23IL0064 and
23IL0075 in different buffers as determined by differential
scanning calorimetry (at 1 mg/mL). Scanning was performed at
1.degree. C./min, starting at 30.degree. C. Buffer 23IL0064
23IL0075 25 mM acetate; pH 5.5; 50 mM NaCl 55.5 58.0 25 mM acetate;
pH 5.5; 250 mM NaCl 52.9 55.5 25 mM MES; pH 6.0; 50 mM NaCl 56.2
58.8 25 mM MES; pH 6.0; 250 mM NaCl 53.2 55.7 25 mM hepes; pH 7.0;
50 mM NaCl 56.7 59.3 25 mM hepes; pH 7.0; 250 mM NaCl 53.7 56.2 25
mM Tris; pH 7.5; 50 mM NaCl 56.3 58.7 25 mM Tris; pH 7.5; 250 mM
NaCl 53.5 56.1
TABLE-US-00034 TABLE 34 Melting temperatures for 23IL0064 and
23IL0075 in buffers as determined by thermal shift assay (at 0.1
mg/mL). Buffer 23IL0064 23IL0075* 23IL0075* 20 mM Histidine pH6.5;
54.5 57.0 57.0 50 mM NaCl 20 mM Histidine pH6.5 56.6 59.2 59.3 20
mM Histidine pH6.5; 7 5% 57.9 60.7 60.6 mannitol 20 mM hepes pH7;
50 mM 54.8 57.4 57.5 NaCl 20 mM hepes pH7 56.8 59.8 59.9 20 mM
hepes pH7; 7.5% 58.2 61.1 61.2 mannitol 20 mM hepes pH8; 50 mM 55.0
57.6 57.8 NaCl 20 mM hepes pH8 56.4 59.5 59.4 20 mM hepes pH8; 7.5%
57.6 60.3 60.3 mannitol *Measurements were performed on the 2
batches
TABLE-US-00035 TABLE 35 Design-Expert Numerical Optimization of the
model. The larger the Desirability coefficient the better the
proposal of the optimum. Conc(log10) Tm Number (mM) PH Buffer
(.degree. C.) Desirability Solutions for phosphate 1 1.00 6.00
Phosphate 59.0012 0.895 2 1.04 6.00 Phosphate 58.9419 0.881
Solutions for Acetate 1 1.00 6.00 Acetate 59.2759 0.959 2 1.01 6.08
Acetate 59.2615 0.956 3 1.11 5.88 Acetate 59.1489 0.929 4 1.10 5.80
Acetate 59.1345 0.926 5 1.15 5.72 Acetate 59.0452 0.905 6 1.16 5.68
Acetate 59.0036 0.895 7 1.32 6.09 Acetate 58.9476 0.882 8 1.33 6.15
Acetate 58.9406 0.881 9 1.35 6.21 Acetate 58.9317 0.879 10 1.19
5.56 Acetate 58.8937 0.870 11 1.32 5.89 Acetate 58.8726 0.865 12
1.00 5.00 Acetate 58.77 0.841 13 1.35 5.69 Acetate 58.7182 0.829 14
1.50 6.12 Acetate 58.7181 0.829 15 1.10 5.10 Acetate 58.682 0.820
16 1.16 5.19 Acetate 58.6346 0.809 17 1.57 6.08 Acetate 58.5857
0.798 18 1.44 5.71 Acetate 58.5814 0.797 19 1.35 5.50 Acetate
58.5692 0.794 20 1.55 5.96 Acetate 58.5672 0.793 21 1.6 6.18
Acetate 58.5354 0.786 22 1.70 6.00 Acetate 58.3294 0.738 23 1.62
5.68 Acetate 58.215 0.711 24 1.34 5.01 Acetate 58.0486 0.672 25
1.47 5.20 Acetate 57.9981 0.660 26 1.33 4.96 Acetate 57.9943 0.659
27 1.56 5.35 Acetate 57.9714 0.654 28 1.27 4.83 Acetate 57.9579
0.651 29 1.62 5.41 Acetate 57.9098 0.639 30 1.53 5.16 Acetate
57.8061 0.615 31 1.61 5.29 Acetate 57.7715 0.607 32 1.57 5.20
Acetate 57.7583 0.604 33 1.56 5.18 Acetate 57.7496 0.602 34 1.35
4.79 Acetate 57.7035 0.591 35 1.65 5.24 Acetate 57.61 0.569 36 1.65
5.17 Acetate 57.5141 0.547 37 1.70 5.00 Acetate 57.0985 0.449 37
Solutions found Solutions for Histidine 1 1.01 6.19 Histidine
59.4631 1.000 2 1.00 6.18 Histidine 59.4654 1.000 3 1.02 6.20
Histidine 59.4617 1.000 4 1.01 6.20 Histidine 59.4767 1.000 5 1.01
6.20 Histidine 59.4702 1.000 6 1.01 6.18 Histidine 59.4525 1.000 7
1.01 6.18 Histidine 59.4554 1.000 8 1.00 6.19 Histidine 59.4736
1.000 9 1.02 6.19 Histidine 59.4524 1.000 10 1.02 6.19 Histidine
59.4537 1.000 11 1.01 6.20 Histidine 59.4644 1.000 12 1.02 6.20
Histidine 59.4559 1.000 13 1.01 6.18 Histidine 59.4608 1.000 14
1.02 6.19 Histidine 59.4556 1.000 15 1.00 6.21 Histidine 59.485
1.000 16 1.01 6.18 Histidine 59.4505 1.000 17 1.02 6.19 Histidine
59.4579 1.000 18 1.03 6.21 Histidine 59.4523 1.000 19 1.01 6.18
Histidine 59.4562 1.000 20 1.01 6.21 Histidine 59.4702 1.000 21
1.00 6.19 Histidine 59.4689 1.000 22 1.01 6.20 Histidine 59.4711
1.000 23 1.05 6.21 Histidine 59.4363 0.997 23 Solutions found
Solutions for Succinate 1 1.00 5.94 Succinate 57.8819 0.633 2 1.00
5.94 Succinate 57.8819 0.633 3 1.00 5.94 Succinate 57.8819 0.633 4
1.00 5.93 Succinate 57.8819 0.633 5 1.00 5.95 Succinate 57.8818
0.633 6 1.00 5.92 Succinate 57.8818 0.633 7 1.00 5.96 Succinate
57.8817 0.633 7 Solutions found
TABLE-US-00036 TABLE 36 Optimization results based on obtained
melting temperatures in the screening of a wide range of excipients
in a histidine, acetate or phosphate buffer for the formulation of
23IL0075. The results were ordered from high to low Tm value. The
formulation composition has to be read combining the identity of
the excipients in columns 2 to 4 and the amount of each excipient
in columns 5 to 7. Combination Sugar/ % sugar/ % amino % Tm value
number polyol Detergent Amino acid Buffer polyol acid Detergent
(.degree. C.) 47 Sucrose Tween 20 Glycine HistidinePH6 10.5 0 0
62.97 44 Sucrose Tween 20 Arg/Glu HistidinePH6 10.5 0 0 62.5 5
Mannitol P-F68 Glycine HistidinePH6 5.6 0 0 62.22 41 Sucrose P-F68
Glycine HistidinePH6 10.5 0 0 62.22 11 Mannitol Tween 20 Glycine
HistidinePH6 5.6 0 0 62.18 46 Sucrose Tween 20 Glycine AcetatePH5.5
10.5 0 0 62.16 42 Sucrose P-F68 Glycine PhosphatePH6 6.3 0.91 0
62.13 38 Sucrose P-F68 Arg/Glu HistidinePH6 10.5 0 0 61.9 6
Mannitol P-F68 Glycine PhosphatePH6 3.7 0.77 0 61.86 43 Sucrose
Tween 20 Arg/Glu AcetatePH5.5 10.5 0 0 61.77 40 Sucrose P-F68
Glycine AcetatePH5.5 8.8 0.37 0 61.74 22 Sorbitol P-F68 Glycine
AcetatePH5.5 3.9 0.69 0 61.7 48 Sucrose Tween 20 Glycine
PhosphatePH6 9.2 0.28 0 61.67 4 Mannitol P-F68 Glycine AcetatePH5.5
4.7 0.37 0 61.58 53 Sucrose Tween 80 Glycine HistidinePH6 10.5 0 0
61.58 54 Sucrose Tween 80 Glycine PhosphatePH6 6.4 0.90 0 61.52 17
Mannitol Tween 80 Glycine HistidinePH6 5.6 0 0 61.44 37 Sucrose
P-F68 Arg/Glu AcetatePH5.5 10.5 0 0 61.43 23 Sorbitol P-F68 Glycine
HistidinePH6 5.6 0 0 61.39 12 Mannitol Tween 20 Glycine
PhosphatePH6 5.6 0 0 61.33 10 Mannitol Tween 20 Glycine
AcetatePH5.5 5.6 0 0 61.2 30 Sorbitol Tween 20 Glycine PhosphatePH6
0.0 2.3 0.0021 61.2 29 Sorbitol Tween 20 Glycine HistidinePH6 5.6 0
0 61.19 50 Sucrose Tween 80 Arg/Glu HistidinePH6 10.5 0 0 61.14 52
Sucrose Tween 80 Glycine AcetatePH5.5 8.4 0.46 0 61.1 2 Mannitol
P-F68 Arg/Glu HistidinePH6 5.6 0 0 61.07 45 Sucrose Tween 20
Arg/Glu PhosphatePH6 10.5 0 0 61.01 18 Mannitol Tween 80 Glycine
PhosphatePH6 5.6 0 0 60.93 8 Mannitol Tween 20 Arg/Glu HistidinePH6
5.6 0 0 60.88 28 Sorbitol Tween 20 Glycine AcetatePH5.5 5.6 0 0
60.86 24 Sorbitol P-F68 Glycine PhosphatePH6 3.2 1.00 0 60.84 16
Mannitol Tween 80 Glycine AcetatePH5.5 5.6 0 0 60.73 19 Sorbitol
P-F68 Arg/Glu AcetatePH5.5 5.6 0 0.062 60.71 49 Sucrose Tween 80
Arg/Glu AcetatePH5.5 10.5 0 0 60.68 20 Sorbitol P-F68 Arg/Glu
HistidinePH6 5.6 0 0.036 60.65 39 Sucrose P-F68 Arg/Glu
PhosphatePH6 10.5 0 0 60.5 1 Mannitol P-F68 Arg/Glu AcetatePH5.5
5.6 0 0 60.43 34 Sorbitol Tween 80 Glycine AcetatePH5.5 3.9 0.69 0
60.29 26 Sorbitol Tween 20 Arg/Glu HistidinePH6 5.6 0 0.0004 60.27
35 Sorbitol Tween 80 Glycine HistidinePH6 5.6 0 0 60.18 14 Mannitol
Tween 80 Arg/Glu HistidinePH6 5.6 0 0 60.17 3 Mannitol P-F68
Arg/Glu PhosphatePH6 5.5 0.06 + 0.05 0 60.15 25 Sorbitol Tween 20
Arg/Glu AcetatePH5.5 5.6 0 0.0017 60.04 7 Mannitol Tween 20 Arg/Glu
AcetatePH5.5 5.6 0 0 59.98 51 Sucrose Tween 80 Arg/Glu PhosphatePH6
10.5 0 0 59.98 9 Mannitol Tween 20 Arg/Glu PhosphatePH6 5.6 0 0
59.86 21 Sorbitol P-F68 Arg/Glu PhosphatePH6 5.6 0 0.106 59.73 13
Mannitol Tween 80 Arg/Glu AcetatePH5.5 5.6 0 0 59.54 15 Mannitol
Tween 80 Arg/Glu PhosphatePH6 5.6 0 0 59.49 36 Sorbitol Tween 80
Glycine PhosphatePH6 5.6 0 0 59.46 31 Sorbitol Tween 80 Arg/Glu
AcetatePH5.5 5.6 0 0.0027 59.36 32 Sorbitol Tween 80 Arg/Glu
HistidinePH6 4.9 0.34 + 0.29 0.0009 59.34 27 Sorbitol Tween 20
Arg/Glu PhosphatePH6 5.6 0 0.0039 59.17 33 Sorbitol Tween 80
Arg/Glu PhosphatePH6 5.6 0 0.0049 58.59
TABLE-US-00037 TABLE 37 Comparison of the aggregation onset
temperatures and maximum scatter reached for 23IL0075 in 3 buffers
(250 .mu.g/mL), as measured by elastic light scattering.
(Temperature interval: 45-95.degree. C., temperature gradient:
2.degree. C./min, data pitch: 1.degree. C., wavelength (ex/em): 500
nm, band width (ex/em): 3 nm.) Aggregation onset Maximum
temperature scatter reached 10 mM phosphate pH 6 52.1.degree. C.
Out of scale 10 mM acetate pH 6 51.0.degree. C. Out of scale 10 mM
histidine pH 6 52.7.degree. C. 435 abs
TABLE-US-00038 TABLE 38 List of buffers tested in freeze/thaw and
stir stress study of 23IL0075 (10 mg/mL) No. Buffer 1 10 mM Acetate
pH 5.5 5.6% mannitol 0.0025% Tween 80 2 10 mM Acetate pH 5.5 5.6%
mannitol 0.005% Tween 80 3 10 mM Acetate pH 5.5 5.6% mannitol 0.05%
P-F68 4 10 mM Acetate pH 5.5 5.6% mannitol 0.1% P-F68 5 10 mM
Histidine pH 6.0 5.6% mannitol 0.0025% Tween 80 6 10 mM Histidine
pH 6.0 5.6% mannitol 0.005% Tween 80 7 10 mM Histidine pH 6.0 5.6%
mannitol 0.05% P-F68 8 10 mM Histidine pH 6.0 5.6% mannitol 0.1%
P-F68 9 10 mM Phosphate pH 6.0 5.6% mannitol 0.0025% Tween 80 10 10
mM Phosphate pH 6.0 5.6% mannitol 0.005% Tween 80 11 10 mM
Phosphate pH 6.0 5.6% mannitol 0.05% P-F68 12 10 mM Phosphate pH
6.0 5.6% mannitol 0.1% P-F68 13 10 mM Acetate pH 5.5 2.8% mannitol
1.15% glycine 0.0025% Tween 80 14 10 mM Acetate pH 5.5 2.8%
mannitol 1.15% glycine 0.005% Tween 80 15 10 mM Acetate pH 5.5 2.8%
mannitol 1.15% glycine 0.05% P-F68 16 10 mM Acetate pH 5.5 2.8%
mannitol 1.15% glycine 0.1% P-F68 17 10 mM Histidine pH 6.0 2.8%
mannitol 1.15% glycine 0.0025% Tween 80 18 10 mM Histidine pH 6.0
2.8% mannitol 1.15% glycine 0.005% Tween 80 19 10 mM Histidine pH
6.0 2.8% mannitol 1.15% glycine 0.05% P-F68 20 10 mM Histidine pH
6.0 2.8% mannitol 1.15% glycine 0.1% P-F68 21 10 mM Phosphate pH
6.0 2.8% mannitol 1.15% glycine 0.0025% Tween 80 22 10 mM Phosphate
pH 6.0 2.8% mannitol 1.15% glycine 0.005% Tween 80 23 10 mM
Phosphate pH 6.0 2.8% mannitol 1.15% glycine 0.05% P-F68 24 10 mM
Phosphate pH 6.0 2.8% mannitol 1.15% glycine 0.1% P-F68
TABLE-US-00039 TABLE 39 Stability study of 23IL0075 in 10 mM
Histidine pH 6.0 with different excipients. The samples were
stressed by 10 .times. freeze/thaw, and were stored at different
temperatures (-70.degree. C., 5.degree. C., 25.degree. C. and
37.degree. C.) for a stability study. The stressed and stability
samples were analyzed using OD measurement, RP-HPLC and SE-HPLC.
Conc. Poloxamer No. (mg/mL) Buffer Mannitol Sucrose Glycine 188
Tween-80 1 25 10 mM L- 5.4% 0.005% histidine, pH 6 2 25 10 mM L-
10.0% 0.005% histidine, pH 6 3 25 10 mM L- 3.5% 3.5% 0.005%
histidine, pH 6 4 25 10 mM L- 2.8% 1.15% 0.005% histidine, pH 6 5
25 10 mM L- 5.4% 0.05% histidine, pH 6 6 25 10 mM L- 10.0% 0.05%
histidine, pH 6 7 25 10 mM L- 3.5% 3.5% 0.05% histidine, pH 6 8 25
10 mM L- 2.8% 1.15% 0.05% histidine, pH 6 9 25 10 mM L- 3.5% 3.5%
histidine, pH 6
TABLE-US-00040 TABLE 40 Samples subjected to shear stress by
stirring. The samples were afterwards analyzed by OD measurements,
RP-HPLC, SE-HPLC and Biacore. No. Conc. Buffer Mannitol Sucrose
Glycine 1 10 mg/mL 10 mM L-histidine, 5.4% pH 6 2 10 mg/mL 10 mM
L-histidine, 10.0% pH 6 3 10 mg/mL 10 mM L-histidine, 3.5% 3.5% pH
6 4 10 mg/mL 10 mM L-histidine, 2.8% 1.15% pH 6
TABLE-US-00041 TABLE 41 Results of SE-HPLC and RP-HPLC
chromatography of 23IL0075 at 25 mg/mL in different candidate
formulation buffers (details see table above) in accelerated
stability study at 25 and 37.degree. C. 3 weeks 37.degree. C. %
postpeak % pre-peak in RP-HPLC 6 weeks 37.degree. C. 6 weeks
25.degree. C. in SE-HPLC (N-terminal % pre-peak* % postpeak** %
pre-peak* % postpeak** Excipients (oligomers)* pyroglutamate)** in
SE-HPLC in RP-HPLC in SE-HPLC in RP-HPLC Mannitol/Tween 80 4.8 6.7
6.9 9.5 0.7 4.9 Sucrose/Tween 80 4.0 6.0 7.1 9.2 0.6 4.1
Mannitol/sucrose/Tween 5.3 5.6 9.5 8.5 0.6 3.8 80
Mannitol/Glycine/Tween 7.2 5.6 12 8.8 0.7 3.9 80 Mannitol/PF 5.0
6.2 12*** 14.7*** 0.8 5.6 Sucrose/PF 3.7 5.8 7.9 9.4 0.6 4.1
Mannitol/Sucrose/PF 4.3 5.4 8.5 8.3 0.7 4.1 Mannitol/Glycine/PF 5.4
5.4 11 8.6 0.8 3.9 Mannitol/Sucrose 3.5 5.2 7.5 8.3 0.8 3.8 *In the
reference sample 0.3 to 0.4% pre-peak in SE-HPLC; **% post-peak in
RP-HPLC was present; ***The sample stressed for 6 weeks at
37.degree. C. in mannitol and Poloxamer 188 was an outlier also
based on the OD500 value that was 0.1 compared to OD500 values
always below 0.01 for all other samples.
TABLE-US-00042 TABLE 42 Stability data of IL6R304 batch CMC-D-0048,
stored at -70.degree. C. Time point (months) Test Method Initial
(0) 3 6 Appearance Clear, colorless solution Clear, colorless
solution Clear, colorless solution A280 10.52 mg/mL 10.38 mg/mL
10.45 mg/mL SEC-HPLC Purity = 99.20% Purity = 98.67% Purity =
98.76% Pre peaks = 0.80% Pre peaks = 1.33% Pre peaks = 1.24% Post
peaks = 0.00% Post peaks = 0.00% Post peaks = 0.00% cIEF Purity =
100.00% Purity = 100.00% Purity = 99.30% Post peak = 0.00% Post
peak = 0.00% Post peak = 0.70% RP-HPLC Purity = 93.90% Purity =
91.93% Purity = 92.8% Pre peak 1 = 0.00% Pre peak 1 = 0.14% Pre
peak 1 = 0.12% Pre peak 2 = 3.20% Pre peak 2 = 3.41% Pre peak 2 =
3.00% Post peak 1 = 0.00% Post peak 1 = 0.00% Post peak 1 = 0.00%
Post peak 2 = 2.60% Post peak 2 = 4.10% Post peak 2 = 3.60% Post
peak 3 = 0.00% Post peak 3 = 0.00% Post peak 3 = 0.00% Post peak 4
= 0.20% Post peak 4 = 0.29% Post peak 4 = 0.32% Post peak 5 = 0.00%
Post peak 5 = 0.14% Post peak 5 = 0.15% Potency 1.256 .+-. 0.084
0.973 .+-. 0.072 1.049 .+-. 0.090 (IL-6R inhibition) Potency 1.044
.+-. 0.094 0.955 .+-. 0.085 0.985 .+-. 0.069 (HSA binding)
TABLE-US-00043 TABLE 43 Stability data of IL6R304 batch CMC-D-0048,
stored at +5.degree. C. Time point (months) Test Method Initial (0)
3 6 Appearance Clear, colorless solution Clear, colorless solution
Clear, colorless solution A280 10.52 mg/mL 10.29 mg/mL 10.33 mg/mL
SEC-HPLC Purity = 99.20% Purity = 98.50% Purity = 98.62% Pre peaks
= 0.80% Pre peaks = 1.50% Pre peaks = 1.38% Post peaks = 0.00% Post
peaks = 0.00% Post peaks = 0.00% cIEF Purity = 100.00% Purity =
100.00% Purity = 99.30% Post peak = 0.00% Post peak = 0.00% Post
peak = 0.70% RP-HPLC Purity = 93.90% Purity = 91.71% Purity =
92.30% Pre peak 1 = 0.00% Pre peak 1 = 0.15% Pre peak 1 = 0.11% Pre
peak 2 = 3.20% Pre peak 2 = 3.53% Pre peak 2 = 3.20% Post peak 1 =
0.00% Post peak 1 = 0.00% Post peak 1 = 0.00% Post peak 2 = 2.60%
Post peak 2 = 4.16% Post peak 2 = 3.90% Post peak 3 = 0.00% Post
peak 3 = 0.00% Post peak 3 = 0.00% Post peak 4 = 0.20% Post peak 4
= 0.29% Post peak 4 = 0.35% Post peak 5 = 0.00% Post peak 5 = 0.15%
Post peak 5 = 0.12% Potency 1.256 .+-. 0.084 0.959 .+-. 0.061 1.015
.+-. 0.076 (IL6R inhibition) Potency 1.044 .+-. 0.094 0.930 .+-.
0.103 0.983 .+-. 0.078 (HSA binding)
TABLE-US-00044 TABLE 44 Stability data of IL6R304 batch CMC-D-0048,
stored at +25.degree. C. Time point (months) Test Method Initial
(0) 3 6 Appearance Clear, colorless solution Clear, colorless
solution Clear, colorless solution A280 10.52 mg/mL 10.29 mg/mL
10.48 mg/mL SEC-HPLC Purity = 99.20% Purity = 97.81% Purity =
97.13% Pre peaks = 0.80% Pre peaks = 1.57% Pre peaks = 1.87% Post
peaks = 0.00% Post peaks = 0.62% Post peaks = 1.00% cIEF Purity =
100.00% Purity = 96.40% Purity = 92.30% Post peak = 0.00% Post peak
= 3.60% Post peak = 7.70% RP-HPLC Purity = 93.90% Purity = 87.77%
Purity = 82.30% Pre peak 1 = 0.00% Pre peak 1 = 0.44% Pre peak 1 =
0.87% Pre peak 2 = 3.20% Pre peak 2 = 4.56% Pre peak 2 = 6.40% Post
peak 1 = 0.00% Post peak 1 = 0.00% Post peak 1 = 1.10% Post peak 2
= 2.60% Post peak 2 = 6.26% Post peak 2 = 8.80% Post peak 3 = 0.00%
Post peak 3 = 0.54% Post peak 3 = 0.86% Post peak 4 = 0.20% Post
peak 4 = 0.30% Post peak 4 = 0.32% Post peak 5 = 0.00% Post peak 5
= 0.13% Post peak 5 = 0.16% Potency 1.256 .+-. 0.084 0.945 .+-.
0.065 0.949 .+-. 0.066 (IL6R inhibition) Potency 1.044 .+-. 0.094
0.967 .+-. 0.095 0.926 .+-. 0.065 (HSA binding)
Sequence CWU 1
1
61245PRTArtificial SequenceNanobody sequence 1Glu Val Gln Leu Val
Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Ser Val Phe Lys Ile Asn 20 25 30Val Met Ala
Trp Tyr Arg Gln Ala Pro Gly Lys Gly Arg Glu Leu Val 35 40 45Ala Gly
Ile Ile Ser Gly Gly Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu65 70 75
80Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala
85 90 95Phe Ile Thr Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp
Gly 100 105 110Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly
Ser Gly Gly 115 120 125Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
Gly Leu Val Gln Pro 130 135 140Gly Asn Ser Leu Arg Leu Ser Cys Ala
Ala Ser Gly Phe Thr Phe Ser145 150 155 160Ser Phe Gly Met Ser Trp
Val Arg Gln Ala Pro Gly Lys Gly Leu Glu 165 170 175Trp Val Ser Ser
Ile Ser Gly Ser Gly Ser Asp Thr Leu Tyr Ala Asp 180 185 190Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Thr Thr 195 200
205Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr
210 215 220Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly
Thr Leu225 230 235 240Val Thr Val Ser Ser 2452375PRTArtificial
SequenceNanobody sequence 2Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Val Phe Lys Ile Asn 20 25 30Val Met Ala Trp Tyr Arg Gln Ala
Pro Gly Lys Gly Arg Glu Leu Val 35 40 45Ala Gly Ile Ile Ser Gly Gly
Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Phe Ile Thr
Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120
125Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
130 135 140Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Ser Val
Phe Lys145 150 155 160Ile Asn Val Met Ala Trp Tyr Arg Gln Ala Pro
Gly Lys Gly Arg Glu 165 170 175Leu Val Ala Gly Ile Ile Ser Gly Gly
Ser Thr Ser Tyr Ala Asp Ser 180 185 190Val Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu 195 200 205Tyr Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr 210 215 220Cys Ala Phe
Ile Thr Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr225 230 235
240Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser
245 250 255Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val 260 265 270Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Phe Thr 275 280 285Phe Ser Ser Phe Gly Met Ser Trp Val Arg
Gln Ala Pro Gly Lys Gly 290 295 300Leu Glu Trp Val Ser Ser Ile Ser
Gly Ser Gly Ser Asp Thr Leu Tyr305 310 315 320Ala Asp Ser Val Lys
Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys 325 330 335Thr Thr Leu
Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala 340 345 350Val
Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly 355 360
365Thr Leu Val Thr Val Ser Ser 370 3753375PRTArtificial
SequenceNanobody sequence 3Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Ser Val Phe Lys Ile Asn 20 25 30Val Met Ala Trp Tyr Arg Gln Ala
Pro Gly Lys Gly Arg Glu Leu Val 35 40 45Ala Gly Ile Ile Ser Gly Gly
Ser Thr Ser Tyr Ala Asp Ser Val Lys 50 55 60Gly Arg Phe Thr Ile Ser
Arg Asp Asn Ala Lys Asn Thr Leu Tyr Leu65 70 75 80Gln Met Asn Ser
Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala 85 90 95Phe Ile Thr
Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly 100 105 110Gln
Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120
125Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro
130 135 140Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr
Phe Ser145 150 155 160Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro
Gly Lys Gly Leu Glu 165 170 175Trp Val Ser Ser Ile Ser Gly Ser Gly
Ser Asp Thr Leu Tyr Ala Asp 180 185 190Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg Asp Asn Ala Lys Thr Thr 195 200 205Leu Tyr Leu Gln Met
Asn Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr 210 215 220Tyr Cys Thr
Ile Gly Gly Ser Leu Ser Arg Ser Ser Gln Gly Thr Leu225 230 235
240Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly Gly Ser Glu Val
245 250 255Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly
Ser Leu 260 265 270Arg Leu Ser Cys Ala Ala Ser Gly Ser Val Phe Lys
Ile Asn Val Met 275 280 285Ala Trp Tyr Arg Gln Ala Pro Gly Lys Gly
Arg Glu Leu Val Ala Gly 290 295 300Ile Ile Ser Gly Gly Ser Thr Ser
Tyr Ala Asp Ser Val Lys Gly Arg305 310 315 320Phe Thr Ile Ser Arg
Asp Asn Ala Lys Asn Thr Leu Tyr Leu Gln Met 325 330 335Asn Ser Leu
Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys Ala Phe Ile 340 345 350Thr
Thr Glu Ser Asp Tyr Asp Leu Gly Arg Arg Tyr Trp Gly Gln Gly 355 360
365Thr Leu Val Thr Val Ser Ser 370 3754385PRTArtificial
SequenceNanobody sequence 4Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30Pro Met Gly Trp Phe Arg Gln Ala
Pro Gly Lys Gly Arg Glu Phe Val 35 40 45Ser Ser Ile Thr Gly Ser Gly
Gly Ser Thr Tyr Tyr Ala Asp Ser Val 50 55 60Lys Gly Arg Phe Thr Ile
Ser Arg Asp Asn Ala Lys Asn Thr Leu Tyr65 70 75 80Leu Gln Met Asn
Ser Leu Arg Pro Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95Ala Ala Tyr
Ile Arg Pro Asp Thr Tyr Leu Ser Arg Asp Tyr Arg Lys 100 105 110Tyr
Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly 115 120
125Gly Gly Ser Gly Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly
130 135 140Gly Leu Val Gln Pro Gly Asn Ser Leu Arg Leu Ser Cys Ala
Ala Ser145 150 155 160Gly Phe Thr Phe Ser Ser Phe Gly Met Ser Trp
Val Arg Gln Ala Pro 165 170 175Gly Lys Gly Leu Glu Trp Val Ser Ser
Ile Ser Gly Ser Gly Ser Asp 180 185 190Thr Leu Tyr Ala Asp Ser Val
Lys Gly Arg Phe Thr Ile Ser Arg Asp 195 200 205Asn Ala Lys Thr Thr
Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu 210 215 220Asp Thr Ala
Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg Ser225 230 235
240Ser Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly
245 250 255Gly Gly Ser Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln 260 265 270Pro Gly Gly Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe 275 280 285Ser Ser Tyr Pro Met Gly Trp Phe Arg Gln
Ala Pro Gly Lys Gly Arg 290 295 300Glu Phe Val Ser Ser Ile Thr Gly
Ser Gly Gly Ser Thr Tyr Tyr Ala305 310 315 320Asp Ser Val Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn 325 330 335Thr Leu Tyr
Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala Val 340 345 350Tyr
Tyr Cys Ala Ala Tyr Ile Arg Pro Asp Thr Tyr Leu Ser Arg Asp 355 360
365Tyr Arg Lys Tyr Asp Tyr Trp Gly Gln Gly Thr Leu Val Thr Val Ser
370 375 380Ser3855386PRTArtificial SequenceNanobody sequence 5Glu
Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10
15Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Leu Pro
20 25 30Ala Ser Gly Asn Ile Phe Asn Leu Leu Thr Ile Ala Trp Tyr Arg
Gln 35 40 45Ala Pro Gly Lys Gly Arg Glu Leu Val Ala Thr Ile Asn Ser
Gly Ser 50 55 60Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr
Ile Ser Arg65 70 75 80Asp Asn Ser Lys Lys Thr Leu Tyr Leu Gln Met
Asn Ser Leu Arg Pro 85 90 95Glu Asp Thr Ala Val Tyr Tyr Cys Gln Thr
Ser Gly Ser Gly Ser Pro 100 105 110Asn Phe Trp Gly Gln Gly Thr Leu
Val Thr Val Ser Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Ser
Glu Val Gln Leu Val Glu Ser Gly Gly Gly 130 135 140Leu Val Gln Pro
Gly Asn Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly145 150 155 160Phe
Thr Phe Ser Ser Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170
175Lys Gly Leu Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr
180 185 190Leu Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg
Asp Asn 195 200 205Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu
Arg Pro Glu Asp 210 215 220Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly
Ser Leu Ser Arg Ser Ser225 230 235 240Gln Gly Thr Leu Val Thr Val
Ser Ser Gly Gly Gly Gly Ser Gly Gly 245 250 255Gly Ser Glu Val Gln
Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro 260 265 270Gly Gly Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Arg Thr Leu Ser 275 280 285Ser
Tyr Ala Met Gly Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 290 295
300Phe Val Ser Arg Ile Ser Gln Gly Gly Thr Ala Ile Tyr Tyr Ala
Asp305 310 315 320Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn
Ser Lys Asn Thr 325 330 335Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro
Glu Asp Thr Ala Val Tyr 340 345 350Tyr Cys Ala Lys Asp Pro Ser Pro
Tyr Tyr Arg Gly Ser Ala Tyr Leu 355 360 365Leu Ser Gly Ser Tyr Asp
Ser Trp Gly Gln Gly Thr Leu Val Thr Val 370 375 380Ser
Ser3856386PRTArtificial SequenceNanobody sequence 6Glu Val Gln Leu
Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly1 5 10 15Ser Leu Arg
Leu Ser Cys Ala Ala Ser Gly Arg Ile Phe Ser Leu Pro 20 25 30Ala Ser
Gly Asn Ile Phe Asn Leu Leu Thr Ile Ala Trp Tyr Arg Gln 35 40 45Ala
Pro Gly Lys Gly Arg Glu Leu Val Ala Thr Ile Asn Ser Gly Ser 50 55
60Arg Thr Tyr Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg65
70 75 80Asp Asn Ser Lys Lys Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg
Pro 85 90 95Glu Asp Thr Ala Val Tyr Tyr Cys Gln Thr Ser Gly Ser Gly
Ser Pro 100 105 110Asn Phe Trp Gly Gln Gly Thr Leu Val Thr Val Ser
Ser Gly Gly Gly 115 120 125Gly Ser Gly Gly Gly Ser Glu Val Gln Leu
Val Glu Ser Gly Gly Gly 130 135 140Leu Val Gln Pro Gly Asn Ser Leu
Arg Leu Ser Cys Ala Ala Ser Gly145 150 155 160Phe Thr Phe Ser Ser
Phe Gly Met Ser Trp Val Arg Gln Ala Pro Gly 165 170 175Lys Gly Leu
Glu Trp Val Ser Ser Ile Ser Gly Ser Gly Ser Asp Thr 180 185 190Leu
Tyr Ala Asp Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn 195 200
205Ala Lys Thr Thr Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp
210 215 220Thr Ala Val Tyr Tyr Cys Thr Ile Gly Gly Ser Leu Ser Arg
Ser Ser225 230 235 240Gln Gly Thr Leu Val Thr Val Ser Ser Gly Gly
Gly Gly Ser Gly Gly 245 250 255Gly Ser Glu Val Gln Leu Leu Glu Ser
Gly Gly Gly Leu Val Gln Pro 260 265 270Gly Gly Ser Leu Arg Leu Ser
Cys Ala Ala Ser Gly Arg Thr Leu Ser 275 280 285Ser Tyr Ala Met Gly
Trp Phe Arg Gln Ala Pro Gly Lys Gly Arg Glu 290 295 300Phe Val Ala
Arg Ile Ser Gln Gly Gly Thr Ala Ile Tyr Tyr Ala Asp305 310 315
320Ser Val Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr
325 330 335Leu Tyr Leu Gln Met Asn Ser Leu Arg Pro Glu Asp Thr Ala
Val Tyr 340 345 350Tyr Cys Ala Lys Asp Pro Ser Pro Tyr Tyr Arg Gly
Ser Ala Tyr Leu 355 360 365Leu Ser Gly Ser Tyr Asp Ser Trp Gly Gln
Gly Thr Leu Val Thr Val 370 375 380Ser Ser385
* * * * *