U.S. patent application number 13/896622 was filed with the patent office on 2013-11-21 for high-concentration monoclonal antibody formulations.
This patent application is currently assigned to Genentech, Inc.. The applicant listed for this patent is GENENTECH, INC.. Invention is credited to Nicholas J. Armstrong, Mayumi N. Bowen, Yuh-Fun Maa.
Application Number | 20130309226 13/896622 |
Document ID | / |
Family ID | 48471141 |
Filed Date | 2013-11-21 |
United States Patent
Application |
20130309226 |
Kind Code |
A1 |
Armstrong; Nicholas J. ; et
al. |
November 21, 2013 |
HIGH-CONCENTRATION MONOCLONAL ANTIBODY FORMULATIONS
Abstract
The present application discloses high-concentration monoclonal
antibody formulations suitable for subcutaneous administration,
e.g. via a pre-filled syringe. In particular, it discloses a
formulation comprising a spray dried monoclonal antibody at a
concentration of about 200 mg/mL or more suspended in a non-aqueous
suspension vehicle where the viscosity of the suspension vehicle is
less than about 20 centipoise. Also disclosed are: a subcutaneous
administration device with the formulation therein, a method of
making the formulation, a method of making an article of
manufacture comprising the suspension formulation, use of the
formulation in the preparation of a medicament, and a method of
treating a patient with the formulation.
Inventors: |
Armstrong; Nicholas J.;
(South San Francisco, CA) ; Bowen; Mayumi N.; (El
Cerrito, CA) ; Maa; Yuh-Fun; (Millbrae, CA) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
GENENTECH, INC. |
South San Francisco |
CA |
US |
|
|
Assignee: |
Genentech, Inc.
South San Francisco
CA
|
Family ID: |
48471141 |
Appl. No.: |
13/896622 |
Filed: |
May 17, 2013 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61649146 |
May 18, 2012 |
|
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|
Current U.S.
Class: |
424/133.1 ;
424/141.1; 424/142.1; 424/143.1; 424/144.1; 424/145.1 |
Current CPC
Class: |
A61P 29/00 20180101;
C07K 16/32 20130101; A61P 27/02 20180101; C07K 16/22 20130101; A61K
9/0019 20130101; A61K 39/3955 20130101; A61P 37/00 20180101; A61K
9/1694 20130101; C07K 2317/21 20130101; A61K 39/39541 20130101;
A61P 35/00 20180101; A61K 9/10 20130101; C07K 16/2887 20130101;
C07K 16/40 20130101; A61P 35/02 20180101; A61K 9/1623 20130101 |
Class at
Publication: |
424/133.1 ;
424/141.1; 424/142.1; 424/144.1; 424/143.1; 424/145.1 |
International
Class: |
A61K 9/16 20060101
A61K009/16; A61K 39/395 20060101 A61K039/395 |
Claims
1. A suspension formulation comprising a spray dried monoclonal
antibody at a concentration of about 200 mg/mL or more suspended in
a non-aqueous suspension vehicle, wherein the viscosity of the
suspension vehicle is less than about 20 centipoise.
2. The formulation of claim 1 wherein the viscosity of the
suspension vehicle is less than about 10 centipoise.
3. The formulation of claim 2 wherein the viscosity of the
suspension vehicle is less than about 5 centipoise.
4. The formulation of claim 1 wherein the injection glide force of
the formulation is about 20 newton or less.
5. The formulation of claim 4 wherein the injection glide force of
the formulation is about 15 newton or less.
6. The formulation of claim 1 wherein the average particle size in
the formulation is from about 2 microns to about 30 microns.
7. The formulation of claim 6 wherein the average particle size in
the formulation is from about 2 microns to about 10 microns.
8. The formulation of claim 1 wherein the antibody concentration in
the formulation is from about 200 mg/mL to about 500 mg/mL.
9. The formulation of claim 8 wherein the antibody concentration in
the formulation is from about 200 mg/mL to about 350 mg/mL.
10. The formulation of claim 1 further comprising a saccharide.
11. The formulation of claim 10 wherein the saccharide is trehalose
or sucrose.
12. The formulation of claim 10 wherein the molar ratio of
saccharide: monoclonal antibody is from about 50 to about
400:1.
13. The formulation of claim 12 wherein the molar ratio of
saccharide: monoclonal antibody is from about 100 to about
250:1.
14. The formulation of claim 1 further comprising a surfactant.
15. The formulation of claim 14 wherein the surfactant is
polysorbate 20 or polysorbate 80.
16. The formulation of claim 1 which is suitable for subcutaneous
administration.
17. The formulation of claim 1 wherein the monoclonal antibody is a
full length monoclonal antibody.
18. The formulation of claim 17 wherein the monoclonal antibody is
a human IgG1.
19. The formulation of claim 1 wherein the monoclonal antibody is a
chimeric, humanized, or human antibody.
20. The formulation of claim 1 wherein the monoclonal antibody
binds an antigen selected from the group consisting of: CD20, HER2,
VEGF, IL6R, beta7, Abeta, HER3, EGFR, and M1'.
21. The formulation of claim 20 wherein the antibody is rituximab,
trastuzumab, or bevacizumab.
22. The formulation of claim 1 wherein the non-aqueous suspension
vehicle comprises propylene glycol dicarprylate/dicaprate, benzyl
benzoate, ethyl lactate, or mixtures thereof.
23. The formulation of claim 22 wherein the non-aqueous suspension
vehicle comprises ethyl lactate.
24. The formulation of claim 22 wherein the non-aqueous suspension
vehicle comprises a mixture of propylene glycol
dicarprylate/dicaprate and ethyl lactate.
25. A subcutaneous administration device with the formulation of
claim 1 therein.
26. The device of claim 25 which comprises a pre-filled
syringe.
27. A method of making a suspension formulation comprising
suspending a spray dried monoclonal antibody in a non-aqueous
suspension vehicle with a viscosity less than about 20 centipoise,
wherein the monoclonal antibody concentration in the suspension
formulation is about 200 mg/mL or more.
28. A method of making an article of manufacture comprising filling
a subcutaneous administration device with the formulation of claim
1.
29. A suspension formulation comprising a spray dried full length
human IgG1 monoclonal antibody at a concentration from about 200
mg/mL to about 400 mg/mL suspended in a non-aqueous suspension
vehicle with a viscosity less than about 20 centipoise, wherein the
formulation has an average particle size from about 2 microns to
about 10 microns, and injection glide force less than about 15
newton.
30. The formulation of claim 29 which further comprises saccharide
wherein the molar ratio of saccharide: monoclonal antibody is from
about 100 to about 250:1.
31. The formulation of claim 29 wherein the antibody is rituximab,
trastuzumab, or bevacizumab.
32. The formulation of claim 1 for use in treating a disease or
disorder in a patient.
33. Use of the formulation of claim 1 in the preparation of a
medicament for treating a patient in need of treatment with the
monoclonal antibody in the formulation.
34. A method of treating a patient comprising administering the
formulation of claim 1 to a patient in need of treatment with the
monoclonal antibody in the formulation.
35. The method of claim 34 wherein the formulation is administered
subcutaneously to the patient.
36. The method of claim 34 wherein the formulation is administered
by a pre-filled syringe containing the formulation therein.
Description
[0001] This non-provisional application filed under 37 CFR
.sctn.1.53(b), claims the benefit under 35 USC .sctn.119(e) of U.S.
Provisional Application Ser. No. 61/649,146, filed on May 18, 2012,
which is incorporated by reference in entirety.
FIELD OF THE INVENTION
[0002] The present invention concerns high-concentration monoclonal
antibody formulations suitable for subcutaneous administration,
e.g. via a pre-filled syringe. In particular, the invention
concerns a formulation comprising a spray dried monoclonal antibody
at a concentration of about 200 mg/mL or more suspended in a
non-aqueous suspension vehicle, wherein the viscosity of the
suspension vehicle is less than about 20 centipoise. The invention
also concerns a subcutaneous administration device with the
formulation therein, a method of making the suspension formulation,
a method of making an article of manufacture comprising the
suspension formulation, use of the suspension formulation in the
preparation of a medicament, and a method of treating a patient
with the suspension formulation.
BACKGROUND OF THE INVENTION
[0003] Outpatient administration of high-dose monoclonal antibodies
(several mg per kg) via subcutaneous (SC) injection is a preferred
form of delivery for treating chronic conditions (Stockwin and
Holmes, Expert Opin. Biol Ther 3:1133-1152 (2003); Shire et al., J.
Pharm. Sci 93:1390-1402 (2004)). The subcutaneous route of
administration that requires injections using syringes,
auto-injectors, or other devices generally restricts product
formulation with regards to injection volume and solution
viscosity, and device functionalities in terms of injection force
and time. To deliver high-dose of monoclonal antibody with
limitations of injection time, volume, and force, a
high-concentration monoclonal antibody formulation (100 mg/mL or
greater) is required for subcutaneous administration (Stockwin and
Holmes, Expert Opin. Biol Ther 3:1133-1152 (2003); Shire et al., J
Pharm. Sci 93:1390-1402 (2004)). A potential challenge in the
development of high protein concentration formulations is
concentration-dependent solution viscosity. Injection force (or
glide force) is a complex factor influenced by solution viscosity,
the size of the needle (i.e., needle gauge), and surface tension of
container/closure. Smaller needles, e.g., .gtoreq.26 gauge, will
pose less pain sensation to the patients. Overcashier and
co-workers established a viscosity-glide force relationship as a
function of needle gauge based on Hagen-Poiseuille Equation
(Overcashier et al., Am. Pharm Rev. 9(6):77-83 (2006)). With a
27-gauge thin walled (TW) needle (ID, min.: 0.241 mm), the liquid
viscosity should be maintained below 20 centipoise in order not to
exceed the glide force of 20 newton. Unfortunately, formulation
scientists are constantly challenged against a conflicting reality
with high monoclonal antibody concentration and high solution
viscosity (Shire et al., J Pharm Sci 93:1390-1402 (2004); Lanai et
al., J Pharm Sci 97:4219-4227 (2005)). Another challenge with
liquid formulations at high monoclonal antibody concentration is
protein physical stability. Greater aggregation rates and
undesirable opalescence are generally observed in high monoclonal
antibody concentration liquid solutions (Alford et al., J. Pharm.
Sci 97:3005-3021 (2008); Salinas et al., J. Pharm. Sci 99:82-93
(2010); Sukumar et al., Pharm Res 21:1087-1093 (2004)).
[0004] Different formulation strategies have been attempted to
reduce the viscosity of high-concentration monoclonal antibody
liquid solution by formulating with salt, amino acid, or sugar to
balance repulsive and attractive forces through intermediate ionic
strengths (Sukumar et al., Pharm Res 21:1087-1093 (2004); He et
al., J Pharm Sci 100:1330-1340 (2011)). However, the effectiveness
of these approaches may be limited at monoclonal antibody
concentration beyond 100 mg/mL or due to specific characteristics
of certain monoclonal antibodies. Dani and co-workers applied the
approach of reconstituting spray-dried monoclonal antibody powder
to prepare high monoclonal antibody concentration liquid solution
prior to subcutaneous injection (Dani et al., J Pharm Sci
96:1504-1517 (2007)). This approach can certainly improve the
protein stability in the solid state during the entire shelf life,
however the high viscosity issue still remains because the spray
dried monoclonal antibody powder needs to be reconstituted at high
monoclonal antibody concentration prior to injection. A
powder-based approach emerged recently using monoclonal antibody
crystalline particle suspensions (Yang et al., Proc Natl Acad Sci
100:6934-6939 (2003); Trilisky et al., "Crystallization and
liquid-liquid phase separation of monoclonal antibodies and
Fe-fusion proteins: Screening results," AICHE online publication
DOI 10, 1002/btrp.621 (published by Wiley Online Library) (2011)).
It is based on the perception that viscosity of a crystal
monoclonal antibody suspension may be lower than a liquid
formulation at the same monoclonal antibody concentration. However,
no viscosity or injection force data were presented in these
references and this concept remained speculative. Furthermore,
monoclonal antibody crystallization is not yet a mature process
platform applicable to a wide range of monoclonal antibodies
although some successful examples have been presented (Trilisky et
al., "Crystallization and liquid-liquid phase separation of
monoclonal antibodies and Fc-fusion proteins: Screening results,"
AICHE online publication DOI 10, 1002/btrp.621 (published by Wiley
Online Library) (2011)).
[0005] The present invention represents a different powder-based
concept employing a high-concentration monoclonal antibody powder
suspension in a non-aqueous suspension vehicle. The suspension
approach has been comprehensively reviewed (Floyd and Jain,
"Injectable emulsions and suspensions," In: Pharmaceutical Dosage
Forms Disperse Systems Volume 2 (eds. Lieberman H A, Rieger M M,
Banker G S). Dekker, NY, N.Y., p 261-318 (1996); Akers et al., J
Parent Sci & Techn 41:88-96 (1987)) and has been reported for
microsphere/emulsion suspensions in vegetable oils, such as sesame
oil (Larsen et al., Eur J. Pharm. Sci 29:348-354 (2006); Hirano et
al., J Pharm Sci 71:495-500 (1982)), soybean oil (Salmeron et al.,
Drug Dev Ind Pharm 23:133-136 (1997); Karasulu et al., Drug Dev
14:225-233 (2007)), and peanut oil (Santucci et al., J Contr Rel
42:157-164 (1996)) as parenteral injectables. The physical and
chemical forces influencing the properties of non-aqueous
suspensions can be quite different from those of aqueous suspension
due to the absence of electrical effects associated with the DLVO
theory (van der Waals attraction and electrostatic repulsion as the
result of double layer of counterions).
[0006] Pena and co-workers (Pena et al., Intl J Pharm 113:89-96
(1995)) reported rheological characterization of excipient-free
bovine somatotropin (rbSt) powder (lyophilized or spray-dried)
suspension in caprylic/capric triglyceride (MIGLYOL 812.RTM.) oil
with or without polysorbate 80. RbSt is a 191-amino acid peptide
with a molecular weight of 22,000 daltons. Pena et al. determined
that a network formed among drug particle, polysorbate 80, and
MIGLYOL 812.RTM., and a higher viscosity was observed with
increasing polysorbate 80 and powder concentrations. These studies
also found that particle shape/morphology played an important role
in suspension viscosity. The smaller spherical (more densely
packed) spray-dried particles resulted in more viscous suspensions
than the lyophilized counterpart which displayed larger irregular
shaped flakes.
[0007] The non-aqueous powder-based approach for high concentration
monoclonal antibody concentration suspensions remains unexplored.
Studies with the small rbSt peptide in Pena et al. would not
predict the ability to effectively formulate a large tetrameric
monoclonal antibody (about 150,000 daltons). In addition, the oil
vehicles used by Pena et al. were too viscous to be considered for
use in pre-filled syringe administration. The viscosity of MIGLYOL
8120, sesame oil, soybean oil, peanut oil are .about.30 centipoise
(cP) at 25.degree. C., 43 cP at 25.degree. C., 50 cP at 25.degree.
C. and 35 cP at 37.degree. C., respectively. In addition, Pena et
al. determined the suspension performance of spray dried powder was
inferior to lyophilized counterpart.
[0008] Publications describing monoclonal antibody formulations
include: U.S. Pat. No. 6,284,282 (Maa et al.);
[0009] U.S. Pat. Nos. 6,267,958 and 6,685,940 (Andya et al.); U.S.
Pat. No. 6,171,586 (Lam et al.); U.S. Pat. Nos. 6,875,432 and
7,666,413 (Liu et al.); WO2006/044908 (Andya et al.);
US-2011-0076273-A1 (Adler et al.); US 2011/0044977 and WO
2011/012637 (Adler et al.); US 2009/0226530A1 (Lassner et al.);
US-A 2003/0190316 (Kakuta et al.); US-A 2005/0214278 and US-A
2005/0118163 (Mizushima et al.); US-A 2009/0291076 (Morichika et
al.); and US-A 2010/0285011 (Imaeda et al.)
SUMMARY OF THE INVENTION
[0010] The objectives of the present study were to: (1) identify
process parameters that dictate suspension performance; (2) assess
the feasibility of establishing monoclonal antibody powder
suspensions (i.e. .gtoreq.250 mg monoclonal antibody/mL) with
acceptable injectability (i.e. injection force .ltoreq.20 N through
27-gauge thin-walled (TW) needle) and physical suspension
stability; and/or (3) understand the mechanism of suspension
performance. To prepare monoclonal antibody powders, spray drying
was used. Spray drying is a mature, scalable, and efficient
manufacturing process. The short-term effect of spray drying on
monoclonal antibody was studied at accelerated temperature. An
important criterion for suspension vehicle selection was that the
viscosity of the suspension vehicle be below 10 centipoise (cP).
The three model suspension vehicles, propylene glycol
dicaprylate/dicaprate, benzyl benzoate, and ethyl lactate, tested
in this study have low viscosity and met this requirement.
[0011] Inverse gas chromatography (IGC) has been used for surface
energy analysis (SEA) (Newell et al., Pharm. Res 18:662-666 (2001);
Grimsey et al., J. Pharm. Sci 91:571-583 (2002); Newell and
Buckton, Pharm Res 21:1440-1444 (2004); Saleem and Smyth, Drug
Devel & Ind Pharm 34:1002-1010 (2008); Panzer and Schreiber,
Macromolecules 25:3633-3637 (1992)). In IGC, a probe is injected
into a column packed with the powder of interest (stationary phase)
and the time required for the probe to pass through the column (4)
is a measure of the magnitude of the interaction between the probe
and the stationary phase. Surface energy can normally be divided
into polar and dispersive (non-polar) components. Thus, the use of
non-polar (alkanes) and polar (electron acceptor-donor or acid-base
solvents) probes allowed these two surface energy components to be
quantified. Surface energies of the spray-dried particles may serve
as a more direct and relevant indicator to suspension performance
than other particle characteristics. Another parameter is heat of
sorption which is a direct measure of the strength of the
interactions between a solid and gas molecules adsorbed on the
surface (Thielmann F., "Inverse gas chromatography:
Characterization of alumina and related surfaces," In "Encyclopedia
of Surface and Colloid Science Volume 4 (edit by P. Somasundaran).
CRC Press, Boca Raton, Fla., p 3009-3031 (2006); Thielmann and
Butler, "Heat of sorption on microcrystalline cellulose by pulse
inverse gas chromatography at infinite dilution," Surface
Measurement Services Application Note 203
(http://www.thesorptionsolution.com/Information_Application_Notes_IGC.php-
#Aps) (2007)). The IGC method was employed to measure the heat of
sorption between spray dried particles and the suspension vehicle
in this study.
[0012] The experimental data herein demonstrate that the objectives
were achieved, and high-concentration monoclonal antibody
suspension formulations suitable for subcutaneous administration
were developed.
[0013] Thus, in a first aspect, the invention concerns a suspension
formulation comprising a spray dried monoclonal antibody at a
concentration of about 200 mg/mL or more suspended in a non-aqueous
suspension vehicle, wherein the viscosity of the suspension vehicle
is less than about 20 centipoise.
[0014] In another aspect, the invention concerns a suspension
formulation comprising a spray dried full length human IgG1
monoclonal antibody at a concentration from about 200 mg/mL to
about 400 mg/mL suspended in a non-aqueous suspension vehicle with
a viscosity less than about 20 centipoise, wherein the formulation
has an average particle size from about 2 microns to about 10
microns, and injection glide force less than about 15 newton.
[0015] The invention further concerns a subcutaneous administration
device (e.g. a pre-filled syringe) with the formulation
therein.
[0016] In another aspect, the invention concerns a method of making
a suspension formulation comprising suspending a spray dried
monoclonal antibody in a non-aqueous suspension vehicle with a
viscosity less than about 20 centipoise, wherein the antibody
concentration in the suspension formulation is about 200 mg/mL or
more.
[0017] Additionally, the invention provides a method of making an
article of manufacture comprising filling a subcutaneous
administration device with the formulation herein.
[0018] In related aspects, the invention concerns use of the
formulation in the preparation of a medicament for treating a
patient in need of treatment with the monoclonal antibody in the
formulation, as well as a method of treating a patient comprising
administering the formulation to a patient in need of treatment
with the monoclonal antibody in the formulation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] FIG. 1: Antibody stability (as size exclusion chromatography
(SEC) % monomer change from right after spry drying) as a function
of storage time at 40.degree. C. for bevacizumab/trehalose
formulation spray-dried ( ) and freeze dried (.smallcircle.) as
well as for trastuzumab/trehalose formulation spray dried
(.box-solid.) and freeze dried (.quadrature.).
[0020] FIG. 2: The viscosity-powder concentration profiles for
propylene glycol dicaprylate/dicaprate suspensions with three
monoclonal antibody (mAb) powders spray dried with a pilot-scale or
a bench-top spray dryer: bevacizumab by pilot-scale (.diamond.),
bevacizumab by bench-top (.diamond-solid.), trastuzumab by
pilot-scale (.quadrature.), trastuzumab by bench-top (.box-solid.),
rituximab by pilot-scale (.DELTA.), rituximab by bench-top
(.tangle-solidup.), empirical fitting (solid line), and theoretical
fitting from Equation 4 (dash line).
[0021] FIG. 3: The glide force-mAb concentration profiles for
rituximab powder suspension in propylene glycol
dicaprylate/dicaprate (.DELTA.), ethyl lactate (.diamond.), benzyl
benzoate (.smallcircle.) and predicted glide force for mAb liquid
solution extracted from FIG. 4 in Overcashier et al. Am. Pharm Rev.
9(6): 77-83 (2006) (.box-solid.).
[0022] FIG. 4: The profiles of viscosity-mAb concentration for
rituximab powder suspension in propylene glycol
dicaprylate/dicaprate (.DELTA.), in benzyl benzoate (.diamond.),
and in ethyl lactate (.smallcircle.).
[0023] FIG. 5: Particle size distribution of rituximab suspensions
in propylene glycol dicaprylate/dicaprate (.diamond.), in benzyl
benzoate (.quadrature.), and in ethyl lactate (.DELTA.).
[0024] FIGS. 6A-C: Photographs of rituximab suspension at 150 mg/mL
in ethyl lactate after 2-week storage (6A), in ethyl lactate
vortexed after 1-day storage (6B), and in propylene glycol
dicaprylate/dicaprate after 2 weeks storage (6C). (Note: the tape
is not part of the suspension but used for optical focusing during
photo taking.)
[0025] FIGS. 7A and 7B: Rituximab suspensions. FIG. 7A: Particle
size distribution of rituximab suspensions in mixtures of propylene
glycol dicaprylate/dicaprate and ethyl lactate at 100/0
(.diamond.), 75/25 (.box-solid.), 50/50 (.quadrature.), 25/75
(.DELTA.), and 0/100 (.DELTA.). FIG. 7B: Photograph of rituximab
suspension in 75/25 propylene glycol dicaprylate/dicaprate/ethyl
lactate mixture after 2-week storage. (Note: the tape is not part
of the suspension but used for optical focusing during photo
taking.)
[0026] FIGS. 8A-1, 8A-2 and 8B provide the amino acid sequences of
the heavy chain (SEQ ID No. 1) and light chain (SEQ ID No. 2) of
rituximab antibody. Each of the framework regions (FR) and each of
the complementarity determining region (CDR) regions in each
variable region are identified, as are the human gamma 1 heavy
chain constant sequence and human kappa light chain constant
sequence. The variable heavy (VH) region is in SEQ ID No. 3. The
variable light (VL) region is in SEQ ID No. 4. The sequence
identifiers for the CDRs are: CDR H1 (SEQ ID No. 5), CDR H2 (SEQ ID
No. 6), CDR H3 (SEQ ID No. 7), CDR L1 (SEQ ID No. 8), CDR L2 (SEQ
ID No. 9), and CDR L3 (SEQ ID No. 10).
[0027] FIGS. 9A and 9B provide the amino acid sequences of the
heavy chain (SEQ ID No. 11) and light chain (SEQ ID No. 12) of
bevacizumab antibody. The end of each variable region is indicated
with .parallel.. The variable heavy (VH) region is in SEQ ID No.
13. The variable light (VL) region is in SEQ ID No. 14. Each of the
three CDRs in each variable region is underlined. The sequence
identifiers for the CDRs are: CDR H1 (SEQ ID No. 15), CDR H2 (SEQ
ID No. 16), CDR H3 (SEQ ID No. 17), CDR L1 (SEQ ID No. 18), CDR L2
(SEQ ID No. 19), and CDR L3 (SEQ ID No. 20).
[0028] FIGS. 10A and 10B provide the amino acid sequences of the
heavy chain (SEQ ID No. 21) and light chain (SEQ ID No. 22) of
trastuzumab antibody. The end of each variable region is indicated
with .parallel.. The variable heavy (VH) region is in SEQ ID No.
23. The variable light (VL) region is in SEQ ID No. 24. Each of the
three CDRs in each variable region is boxed. The sequence
identifiers for the CDRs are: CDR H1 (SEQ ID No. 25), CDR H2 (SEQ
ID No. 26), CDR H3 (SEQ ID No. 27), CDR L1 (SEQ ID No. 28), CDR L2
(SEQ ID No. 29), and CDR L3 (SEQ ID No. 30).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Definitions
[0029] The term "pharmaceutical formulation" refers to a
preparation which is in such form as to permit the biological
activity of the active agent (e.g. monoclonal antibody) to be
effective, and which contains no additional components which are
unacceptably toxic to a subject to which the formulation would be
administered. Such formulations are sterile. In one embodiment, the
pharmaceutical formulation is suitable for subcutaneous
administration.
[0030] "Pharmaceutically acceptable" with respect to an excipient
in a pharmaceutical formulation means that the excipient is
suitable for administration to a human patient.
[0031] A "sterile" formulation is asceptic or free from all living
microorganisms and their spores.
[0032] "Subcutaneous administration" refers to administration (of a
formulation) under the skin of a subject or patient.
[0033] A "stable" formulation is one in which the active agent
(e.g. monoclonal antibody) therein essentially retains its physical
stability and/or chemical stability and/or biological activity upon
suspension and/or storage. Preferably, the formulation essentially
retains its physical and chemical stability, as well as its
biological activity upon suspension and storage. The storage period
is generally selected based on the intended shelf-life of the
formulation. Various analytical techniques for measuring protein
stability are available in the art and are reviewed in Peptide and
Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker,
Inc., New York, N.Y., Pubs. (1991); and Jones, A. Adv. Drug
Delivery Rev. 10: 29-90 (1993), for example. In one embodiment,
stability of the suspension formulation is assessed around the time
the spray dried particles are suspended in the vehicle to produce
the suspension formulation. In one embodiment, stability can be
evaluated when the formulation is held at a selected temperature
for a selected time period. In one embodiment, monoclonal antibody
stability is assessed by size distribution (percentage monomer,
aggregation, and/or fragmentation) before and after spray drying
(e.g. before and after spray drying over 3-month storage under the
accelerated temperature of 40.degree. C.). In one embodiment, size
distribution is assessed using size exclusion chromatography-high
performance liquid chromatography (SEC-HPLC). In one embodiment,
the percentage monomer loss (as measured by SEC-HPLC) over 3 months
is less than about 10%, for example less than 5%, e.g. at
accelerated temperature of 40.degree. C. In one embodiment,
stability is assessed by evaluating suspension physical stability,
e.g. visual inspection of settling and/or particle sedimentation
rate.
[0034] "Spray drying" refers to the process of atomizing and drying
a liquid or slurry comprising a protein or monoclonal antibody
using gas (usually air or nitrogen) at a temperature above ambient
temperature so as to produce dry powder particles comprising the
protein or monoclonal antibody. During the process, liquid
evaporates and dry particles form. In one embodiment, the spray
drying is performed using a spray dryer, e.g. which has an air
inlet temperature from about 100.degree. C. to about 220.degree. C.
and an air outlet temperature from about 50.degree. C. to about
100.degree. C. Particles can be separated from the gas by various
methods such as cyclone, high pressure gas, electrostatic charge,
etc. This definition of spray drying herein expressly excludes
freeze drying or crystallizing the monoclonal antibody.
[0035] A "dry" particle, protein, or monoclonal antibody herein has
been subjected to a drying process such that its water content has
been significantly reduced. In one embodiment, the particle,
protein, or monoclonal antibody has a water content of less than
about 10%, for example less than about 5%, e.g., where water
content is measured by a chemical titration method (e.g. Karl
Fischer method) or a weight-loss method (high-temperature
heating).
[0036] For the purposes herein, a "pre-spray dried preparation"
refers to a preparation of the monoclonal antibody (usually a
recombinantly produced monoclonal antibody which has been subjected
to one or more purification steps) and one or more excipients, such
as stabilizers (e.g. saccharides, surfactants, and/or amino acids)
and, optionally, a buffer. In one embodiment the preparation is in
liquid form. In one embodiment the preparation is frozen.
[0037] A "suspension formulation" is a liquid formulation
comprising solid particles (e.g. spray dried monoclonal antibody
particles) dispersed throughout a liquid phase in which they are
not soluble. In one embodiment, the solid particles in the
suspension formulation have an average particle diameter from about
2 to about 30 microns, e.g. from about 5 to about 10 microns (e.g.
as analyzed by laser diffraction). Optionally, the solid particles
in the suspension formulation have a peak (highest percentage)
particle size of less than about 30 micron, and optionally less
than about 10 microns (e.g. as analyzed by laser diffraction). The
suspension formulation may be prepared by combinding spray dried
monoclonal antibody particles with a non-aqueous suspension
vehicle. In one embodiment, the suspension formulation is adapted
for, or suitable for, subcutaneous administration to a subject or
patient.
[0038] As used herein "non-aqueous suspension vehicle" refers to a
pharmaceutically acceptable liquid which is not water-based and in
which spray dried monoclonal antibody particles can be suspended in
order to generate a suspension formulation. In one embodiment, the
vehicle comprises a liquid lipid or fatty acid ester or alcohol
(e.g. propylene glycol dicaprylate/dicaprate), or other organic
compound such benzyl benzoate or ethyl lactate. The vehicle herein
includes mixtures of two or more liquids, such as a mixture of
propylene glycol dicaprylate/dicaprate and ethyl lactate.
Preferably, the non-aqueous suspension vehicle has a viscosity (at
25.degree. C.) of less than about 20 centipoise (cP), optionally
less than about 10 cP, and, in one embodiment, less than about 5
cP. Examples of non-aqueous suspension vehicles herein include the
vehicles in the Table 1 below:
TABLE-US-00001 TABLE 1 Exemplary Non-Aqueous Suspension Vehicles
and Their Viscosity Vehicle Viscosity (cP) Ethanol 1.3 (25.degree.
C.) Dimethyl sulfoxide 2.0 (20.degree. C.) N-methyl-2-pyrrolidone
1.66 (25.degree. C.) Acetone 0.33 (20.degree. C.) Benzyl benzoate 9
(25.degree. C.) Tetrahydrofurfuryl alcohol 6.2 (25.degree. C.)
dimethyl ether of diethylene glycol 1.2 (15.degree. C.) (Diglym)
Ethyl lactate 2 (20.degree. C.) Ethyl oleate 7.4 (20.degree. C.)
Isopropyl Myristate 5.7 (20.degree. C.) Propylene glycol
dicaprylate/dicaprate 9 (25.degree. C.) (MIGLYOL 840 .RTM.)
[0039] "Viscosity" refers to the measure of the resistance of a
fluid which is being deformed by either shear stress or tensile
stress; it can be evaluated using a viscometer or rheometer. Unless
indicated otherwise, the viscosity measurement (centipoise, cP) is
that at about 25.degree. C. Viscosity as used herein can refer to
that of either the non-aqueous suspension vehicle per se or that of
the suspension formulation.
[0040] "Injectability" refers to the ease with which the suspension
formulation can be administered to a subject. According to one
embodiment of the invention, the injectibility of a given
suspension formulation can be superior to the injectability of a
liquid formulation comprising the same monoclonal antibody
concentration and the same excipient(s) and concentration(s)
thereof. In one embodiment, injectability refers to the injection
glide force.
[0041] "Injection glide force" as used herein refers to the force
required for the injection of a solution at a given injection rate
via a needle of predetermined gauge and length. In one embodiment,
it is evaluated using pre-filled syringe (e.g. 1.0 mL-long syringe
with .ltoreq.25 gauge needle, or preferably .ltoreq.27 gauge
needle) with glide force analyzed and established as a function of
the distance of the plunger rod travelling inside the syringe at a
steady compression rate (e.g. using "Syringe Glide Force
Measurement" as in the Example herein). Time and force required for
a manual injection (or time required for an injection using an
autoinjector) may impact the usability of the product by the
end-user (and thus compliance with the intended use of the
product). In one embodiment, the Hagen-Poiseuille equation is
utilized to estimate the travel (or glide) force (Equation 1).
F = 8 Q .mu. L .pi. ? .times. A ? indicates text missing or
illegible when filed ( Equation 1 ) ##EQU00001##
Q=Volumetric flow rate .mu.=Fluid viscosity L=Needle length
R=Needle inner diameter A=Cross sectional area of syringe plunger
F=Frictionless travel force
[0042] According to Equation 1, the glide force is dependent on a
number of parameters. The only parameter a formulation scientist
can influence is viscosity. All other parameters (needle inner
diameter, needle length, and cross sectional area of syringe
plunger) are determined by the pre-fillable syringe itself.
Formulations with a high viscosity can lead to high injection
forces and long injection times since both parameters are
proportional to viscosity. Generally accepted limits for injection
force and injection time may depend e.g. on the indication and the
dexterity of the patient population. In an embodiment exemplified
herein, the parameters in Equation 1 were:
Q=Volumetric flow rate=0.1 mL/second .mu.=Fluid viscosity=20
centipoise L=Needle length=1.25 cm R=Needle inner diameter=0.0105
cm (27 gauge needle) A=Cross sectional area of syringe
plunger=0.00316 cm.sup.2 F=Frictionless travel
force=16.6.times.10.sup.5 dyne=16.6 newton
[0043] In one embodiment, injection glide force is determined as a
function of monoclonal antibody concentration by injecting 1-mL of
suspension formulation using a 1-mL long syringe through a 27-guage
thin walled (TW) staked needle in 10 seconds.
[0044] In one embodiment the injection glide force of the
suspension formulation is about 20 newtons or less.
[0045] In one embodiment the injection glide force of the
suspension formulation is about 15 newton or less.
[0046] In one embodiment the injection glide force is from about 2
newton to about 20 newton.
[0047] In one embodiment the injection glide force is from about 2
newton to about 15 newton.
[0048] In one embodiment the injection glide force is less than
about 20 newton.
[0049] In one embodiment the injection glide force is less than
about 15 newton.
[0050] As used herein, "buffer" refers to a buffered solution that
resists changes in pH by the action of its acid-base conjugate
components. The buffer of this invention (if used) generally has a
pH from about 4.0 to about 8.0, for example from about 5.0 to about
7.0, e.g. from about 5.8 to about 6.2, and in one embodiment its pH
is about 6.0. Examples of buffers that will control the pH in this
range include acetate, succinate, succinate, gluconate, histidine,
citrate, glycylglycine and other organic acid buffers. In one
embodiment herein, the buffer is a histidine buffer. A buffer is
generally included in the pre-spray dried preparation and may be
present in the suspension formulation prepared therefrom (but is
not required therein).
[0051] A "histidine buffer" is a buffer comprising histidine ions.
Examples of histidine buffers include histidine chloride, histidine
acetate, histidine phosphate, histidine sulfate. In one embodiment,
the histidine buffer is histidine-acetate or histidine-HCl. In one
embodiment, the histidine buffer is at pH 5.5 to 6.5, optionally pH
5.8 to 6.2, e.g. pH 6.0.
[0052] The term "excipient" refers to an agent that may be added to
a preparation or formulation, for example: as a stabilizer, to
achieve a desired consistency (e.g., altering the bulk properties),
and/or to adjust osmolality. Examples of excipients herein include,
but are not limited to, stabilizers, sugars, polyols, amino acids,
surfactants, chelating agents, and polymers.
[0053] A "stabilizer" herein is an excipient, or mixture of two or
more excipients, which stabilizes a pharmaceutical formulation. For
example, the stabilizer can prevent instability due to spray drying
at elevated temperature. Exemplary stabilizers herein include
saccharides, surfactants, and amino acids.
[0054] A "saccharide" herein comprises the general composition
(CH2O)n and derivatives thereof, including monosaccharides,
disaccharides, trisaccharides, polysaccharides, sugar alcohols,
reducing sugars, nonreducing sugars, etc. Examples of saccharides
herein include glucose, sucrose, trehalose, lactose, fructose,
maltose, dextran, glycerin, dextran, erythritol, glycerol,
arabitol, sylitol, sorbitol, mannitol, mellibiose, melezitose,
raffinose, mannotriose, stachyose, maltose, lactulose, maltulose,
glucitol, maltitol, lactitol, iso-maltulose, etc. The preferred
saccharide herein is a nonreducing disaccharide, such as trehalose
or sucrose.
[0055] Herein, a "surfactant" refers to a surface-active agent,
preferably a nonionic surfactant. Examples of surfactants herein
include polysorbate (for example, polysorbate 20 and, polysorbate
80); poloxamer (e.g. poloxamer 188); Triton; sodium dodecyl sulfate
(SDS); sodium laurel sulfate; sodium octyl glycoside; lauryl-,
myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-,
linoleyl- or stearyl-sarcosine; linoleyl-, myristyl-, or
cetyl-betaine; lauroamidopropyl-, cocamidopropyl-,
linoleamidopropyl-, myristamidopropyl-, paImidopropyl-, or
isostearamidopropyl-betaine (e.g. lauroamidopropyl);
myristamidopropyl-, palmidopropyl-, or
isostearamidopropyl-dimethylamine; sodium methyl cocoyl-, or
disodium methyl oleyl-taurate; and the MONAQUAT.TM. series (Mona
Industries, Inc., Paterson, N.J.); polyethyl glycol, polypropyl
glycol, and copolymers of ethylene and propylene glycol (e.g.
Pluronics, PF68 etc); etc. In one embodiment, the surfactant is
polysorbate 20 or polysorbate 80. The surfactant may be included to
prevent or reduce aggregation or denaturation of the monoclonal
antibody in the preparation and/or formulation.
[0056] The term "amino acid" as used herein denotes a
pharmaceutically acceptable organic molecule possessing an amino
moiety located at a-position to a carboxylic group. Examples of
amino acids include: arginine, glycine, ornithine, lysine,
histidine, glutamic acid, asparagic acid, isoleucine, leucine,
alanine, phenylalanine, tyrosine, tryptophane, methionine, serine,
and proline. The amino acid employed is optionally in the L-form.
Examples of amino acids which can be included as stabilizers in the
preparations and/or formulations herein include: histidine,
arginine, glycine, and/or alanine.
[0057] By "isotonic" is meant that the formulation of interest has
essentially the same osmotic pressure as human blood. Isotonic
formulations will generally have an osmotic pressure from about 250
to 350 mOsm. Isotonicity can be measured using a vapor pressure or
ice-freezing type osmometer, for example.
[0058] The term "monoclonal antibody" as used herein refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical and/or bind the same epitope, except for
possible variants that may arise during production of the
monoclonal antibody, such variants generally being present in minor
amounts. In contrast to polyclonal antibody preparations that
typically include different antibodies directed against different
determinants (epitopes), each monoclonal antibody is directed
against a single determinant on the antigen. In addition to their
specificity, the monoclonal antibodies are advantageous in that
they are uncontaminated by other immunoglobulins. The modifier
"monoclonal" indicates the character of the antibody as being
obtained from a substantially homogeneous population of antibodies,
and is not to be construed as requiring production of the antibody
by any particular method. For example, the monoclonal antibodies to
be used in accordance with the present invention may be made by the
hybridoma method first described by Kohler et al., Nature, 256:495
(1975), or may be made by recombinant DNA methods (see, e.g., U.S.
Pat. No. 4,816,567). The "monoclonal antibodies" may also be
isolated from phage antibody libraries using the techniques
described in Clackson et al., Nature, 352:624-628 (1991) and Marks
et al., J. Mol. Biol., 222:581-597 (1991), for example. Specific
examples of monoclonal antibodies herein include chimeric
antibodies, humanized antibodies, and human antibodies.
[0059] A "spray dried" monoclonal antibody has been subjected to
spray drying. The term includes the spray dried monoclonal antibody
in powder form (i.e. prior to suspension) and in liquid form (i.e.
when suspended in the non-aqueous suspension vehicle to form the
suspension formulation).
[0060] The monoclonal antibodies herein specifically include
"chimeric" antibodies (immunoglobulins) in which a portion of the
heavy and/or light chain is identical with or homologous to
corresponding sequences in antibodies derived from a particular
species or belonging to a particular antibody class or subclass,
while the remainder of the chain(s) is identical with or homologous
to corresponding sequences in antibodies derived from another
species or belonging to another antibody class or subclass, so long
as they exhibit the desired biological activity (U.S. Pat. No.
4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA,
81:6851-6855 (1984)). Chimeric antibodies of interest herein
include "primatized" antibodies comprising variable domain
antigen-binding sequences derived from a non-human primate (e.g.
Old World Monkey, such as baboon, rhesus or cynomolgus monkey) and
human constant region sequences (U.S. Pat. No. 5,693,780). An
example of a chimeric antibody herein is rituximab.
[0061] "Humanized" forms of non-human (e.g., murine) antibodies are
chimeric antibodies that contain minimal sequence derived from
non-human immunoglobulin. For the most part, humanized antibodies
are human immunoglobulins (recipient antibody) in which residues
from a hypervariable region of the recipient are replaced by
residues from a hypervariable region of a non-human species (donor
antibody) such as mouse, rat, rabbit or nonhuman primate having the
desired specificity, affinity, and capacity. In some instances,
framework region (FR) residues of the human immunoglobulin are
replaced by corresponding non-human residues. Furthermore,
humanized antibodies may comprise residues that are not found in
the recipient antibody or in the donor antibody. These
modifications are made to further refine antibody performance. In
general, the humanized antibody will comprise substantially all of
at least one, and typically two, variable domains, in which all or
substantially all of the hypervariable regions correspond to those
of a non-human immunoglobulin and all or substantially all of the
FRs are those of a human immunoglobulin sequence, except for FR
substitution(s) as noted above. The humanized antibody optionally
also will comprise at least a portion of an immunoglobulin constant
region, typically that of a human immunoglobulin. For further
details, see Jones et al., Nature 321; 522-525 (1986); Riechmann et
al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.
2:593-596 (1992). Exemplary humanized antibodies herein include
trastuzumab and bevacizumab.
[0062] A "human antibody" herein is one comprising an amino acid
sequence structure that corresponds with the amino acid sequence
structure of an antibody obtainable from a human B-cell. Such
antibodies can be identified or made by a variety of techniques,
including, but not limited to: production by transgenic animals
(e.g., mice) that are capable, upon immunization, of producing
human antibodies in the absence of endogenous immunoglobulin
production (see, e.g., Jakobovits et al., Proc. Natl. Acad. Sci.
USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993);
Bruggermann et al., Year in. Immuno., 7:33 (1993); and U.S. Pat.
Nos. 5,591,669, 5,589,369 and 5,545,807)); selection from phage
display libraries expressing human antibodies (see, for example,
McCafferty et al., Nature 348:552-553 (1990); Johnson et al.,
Current Opinion in Structural Biology 3:564-571 (1993); Clackson et
al., Nature, 352:624-628 (1991); Marks et al., J. Mol. Biol.
222:581-597 (1991); Griffith et al., EMBO J. 12:725-734 (1993);U.S.
Pat. Nos. 5,565,332 and 5,573,905); generation via in vitro
activated B cells (see U.S. Pat. Nos. 5,567,610 and 5,229,275); and
isolation from human antibody producing hybridomas. An example of a
human antibody herein is ofatumumab.
[0063] A "multispecific antibody" herein is an antibody having
binding specificities for two or more different epitopes.
[0064] A "bispecific antibody" is an antibody with binding
specificities for two different epitopes. An example of a
bispecific antibody specifically contemplated herein is HER3/EGFR
Dual Acting Fab (DAF) molecule, such as DL11f comprising human IgG1
heavy chains (US 2010/0255010; WO2010/108127).
[0065] Antibodies herein include "amino acid sequence variants"
with altered antigen-binding or biological activity. Examples of
such amino acid alterations include antibodies with enhanced
affinity for antigen (e.g. "affinity matured" antibodies), and
antibodies with altered Fc region e.g. with altered (increased or
diminished) antibody dependent cellular cytotoxicity (ADCC) and/or
complement dependent cytotoxicity (CDC) (see, for example, WO
00/42072, Presta, L. and WO 99/51642, Iduosogie et al.); and/or
increased or diminished serum half-life (see, for example,
WO00/42072, Presta, L.).
[0066] An "affinity matured variant" has one or more substituted
hypervariable region residues of a parent antibody (e.g. of a
parent chimeric, humanized, or human antibody) which improve
binding of the affinity matured variant.
[0067] The antibody herein may be conjugated with a "heterologous
molecule" for example to increase half-life or stability or
otherwise improve the antibody. For example, the antibody may be
linked to one of a variety of non-proteinaceous polymers, e.g.,
polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes,
or copolymers of polyethylene glycol and polypropylene glycol.
[0068] The antibody herein may be a "glycosylation variant" such
that any carbohydrate attached to its Fc region is altered. For
example, antibodies with a mature carbohydrate structure that lacks
fucose attached to an Fc region of the antibody are described in US
Pat Appl No US 2003/0157108 (Presta, L.). See also US 2004/0093621
(Kyowa Hakko Kogyo Co., Ltd). Antibodies with a bisecting
N-acetylglucosamine (GlcNAc) in the carbohydrate attached to an Fc
region of the antibody are referenced in WO 2003/011878,
Jean-Mairet et al. and U.S. Pat. No. 6,602,684, Umana et al.
Antibodies with at least one galactose residue in the
oligosaccharide attached to an Fc region of the antibody are
reported in WO 1997/30087, Patel et al. See, also, WO 1998/58964
(Raju, S.) and WO 1999/22764 (Raju, S.) concerning antibodies with
altered carbohydrate attached to the Fc region thereof. See also US
2005/0123546 (Umana et al.) describing antibodies with modified
glycosylation.
[0069] The term "hypervariable region" when used herein refers to
the amino acid residues of an antibody that are responsible for
antigen binding. The hypervariable region comprises amino acid
residues from a "complementarity determining region" or "CDR" (e.g.
residues 24-34 (L1), 50-56 (L2) and 89-97 (L3) in the light chain
variable domain and 31-35 (H1), 50-65 (H2) and 95-102 (H3) in the
heavy chain variable domain; Kabat et al., Sequences of Proteins of
Immunological Interest, 5th Ed. Public Health Service, National
Institutes of Health, Bethesda, Md. (1991)) and/or those residues
from a "hypervariable loop" (e.g. residues 26-32 (L1), 50-52 (L2)
and 91-96 (L3) in the light chain variable domain and 26-32 (H1),
53-55 (H2) and 96-101 (H3) in the heavy chain variable domain;
Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)). "Framework" or
"FR" residues are those variable domain residues other than the
hypervariable region residues as herein defined. The CDRs of
rituximab, bevacizumab, and trastuzumab are disclosed in FIGS.
8A-1, 8A-2, 8B, 9A-B, and 10A-B, respectively.
[0070] A "full length antibody" is one which comprises an
antigen-binding variable region as well as a light chain constant
domain (CL) and heavy chain constant domains, CH1, CH2 and CH3. The
constant domains may be native sequence constant domains (e.g.
human native sequence constant domains) or amino acid sequence
variants thereof. Preferably, the full length antibody has one or
more effector functions. In one embodiment, a human IgG heavy chain
Fc region extends from Cys226, or from Pro230, to the
carboxyl-terminus of the heavy chain. However, the C-terminal
lysine (Lys447) of the Fc region may or may not be present. Unless
otherwise specified herein, numbering of amino acid residues in the
Fe region or constant region is according to the EU numbering
system, also called the EU index, as described in Kabat et al.,
Sequences of Proteins of Immunological Interest, 5th Ed. Public
Health Service, National Institutes of Health, Bethesda, Md., 1991.
Rituximab, trastuzumab, and bevacizumab are examples of full length
antibodies.
[0071] A "naked antibody" is a monoclonal antibody that is not
conjugated to a heterologous molecule, such as a cytotoxic moiety,
polymer, or radiolabel. Rituximab, trastuzumab, and bevacizumab are
examples of naked antibodies.
[0072] Antibody "effector functions" refer to those biological
activities attributable to the Fc region (a native sequence Fc
region or amino acid sequence variant Fc region) of an antibody.
Examples of antibody effector functions include C1q binding,
complement dependent cytotoxicity (CDC), Fc receptor binding,
antibody-dependent cell-mediated cytotoxicity (ADCC), etc.
[0073] Depending on the amino acid sequence of the constant domain
of their heavy chains, full length antibodies can be assigned to
different classes. There are five major classes of full length
antibodies: IgA, IgD, IgE, IgG, and IgM, and several of these may
be further divided into "subclasses" (isotypes), e.g., IgG1, IgG2,
IgG3, IgG4, IgA, and IgA2. The heavy chain constant domains that
correspond to the different classes of antibodies are called alpha,
delta, epsilon, gamma, and mu, respectively. The subunit structures
and three-dimensional configurations of different classes of
immunoglobulins are well known. The antibody herein is a human IgG1
according to one embodiment of the invention.
[0074] A "human IgG1" antibody herein refers to full length
antibody comprising human IgG1 heavy chain constant domains.
[0075] The term "recombinant antibody" as used herein, refers to a
monoclonal antibody (e.g. a chimeric, humanized, or human
monoclonal antibody) that is expressed by a recombinant host cell
comprising nucleic acid encoding the monoclonal antibody. Examples
of "host cells" for producing recombinant antibodies include: (1)
mammalian cells, for example, Chinese Hamster Ovary (CHO), COS,
myeloma cells (including Y0 and NS0 cells), baby hamster kidney
(BHK), Hela and Vero cells; (2) insect cells, for example, sf9,
sf21 and Tn5; (3) plant cells, for example plants belonging to the
genus Nicotiana (e.g. Nicotiana tabacum); (4) yeast cells, for
example, those belonging to the genus Saccharomyces (e.g.
Saccharomyces cerevisiae) or the genus Aspergillus (e.g.
Aspergillus niger); (5) bacterial cells, for example Escherichia
coli cells or Bacillus subtilis cells, etc.
[0076] As used herein, "specifically binding" or "binds
specifically to" refers to an antibody selectively or
preferentially binding to an antigen. Preferably the binding
affinity for antigen is of Kd value of 10.sup.-9 mol/l or lower
(e.g. 10.sup.-10 mol/l), preferably with a Kd value of 10.sup.-10
mol/l or lower (e.g. 10.sup.-12 mol/l). The binding affinity is
determined with a standard binding assay, such as surface plasmon
resonance technique (BIACORE.RTM.).
[0077] A "therapeutic monoclonal antibody" is a monoclonal antibody
used for therapy of a human subject. Therapeutic monoclonal
antibodies disclosed herein include: CD20 antibodies for therapy of
B cell malignancies (such as non-Hodgkin's lymphoma or chronic
lymphocytic leukemia) or autoimmune diseases (such as rheumatoid
arthritis and vasculitis); HER2 antibodies for cancer (such as
breast cancer or gastric cancer); VEGF antibodies for treating
cancer, age-related macular degeneration, macular edema, etc.
[0078] For the purposes herein, "rituximab" refers to an antibody
comprising the variable heavy amino acid sequence in SEQ ID No. 3
and variable light amino acid in SEQ ID No. 4, and, optionally, the
heavy chain amino acid sequence in SEQ ID No. 1 and light chain
amino acid sequence in SEQ ID No. 2. This term specifically
includes biosimilar rituximab.
[0079] For the purposes herein, "bevacizumab" refers to an antibody
comprising the variable heavy amino acid sequence in SEQ ID No. 13
and variable light amino acid in SEQ ID No. 14, and, optionally,
the heavy chain amino acid sequence in SEQ ID No. 11 and light
chain amino acid sequence in SEQ ID No. 12. This term specifically
includes biosimilar bevacizumab,
[0080] For the purposes herein, "trastuzumab" refers to an antibody
comprising the variable heavy amino acid sequence in SEQ ID No. 23
and variable light amino acid in SEQ ID No. 24, and, optionally,
the heavy chain amino acid sequence in SEQ ID No. 21 and light
chain amino acid sequence in SEQ ID No. 22. This term specifically
includes biosimilar trastuzumab.
[0081] The monoclonal antibody which is formulated herein is
preferably essentially pure and desirably essentially homogeneous
(i.e. free from contaminating proteins etc). "Essentially pure"
antibody means a composition comprising at least about 90% by
weight of the antibody, based on total weight of the composition,
preferably at least about 95% by weight. "Essentially homogeneous"
antibody means a composition comprising at least about 99% by
weight of antibody, based on total weight of the composition.
II. Monoclonal Antibodies to be Formulated Herein
[0082] Exemplary techniques for producing monoclonal antibodies
which can be formulated according to the present invention follow.
In one embodiment, the antigen to which the antibody binds is a
biologically important protein and administration of the antibody
to a mammal suffering from a disease or disorder can result in a
therapeutic benefit in that mammal. However, antibodies directed
against nonpolypeptide antigens (such as tumor-associated
glycolipid antigens; see U.S. Pat. No. 5,091,178) are also
contemplated.
[0083] Where the antigen is a polypeptide, it may be a
transmembrane molecule (e.g. receptor) or ligand such as a growth
factor. Exemplary antigens include molecules such as renin; a
growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor (TF), and
von Willebrands factor; anti-clotting factors such as Protein C;
atrial natriuretic factor; lung surfactant; a plasminogen
activator, such as urokinase or human urine or tissue-type
plasminogen activator (t-PA); bombesin; thrombin; hemopoietic
growth factor; tumor necrosis factor-alpha and -beta;
enkephalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MIP-1-alpha); a serum albumin such as human serum albumin;
muellerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; a microbial
protein, such as beta-lactamase; DNase; IgE; a cytotoxic
T-lymphocyte associated antigen (CTLA), such as CTLA-4; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors; protein A or D; rheumatoid factors; a
neurotrophic factor such as bone-derived neurotrophic factor
(BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6),
or a nerve growth factor such as NGF-b; platelet-derived growth
factor (PDGF); fibroblast growth factor such as aFGF and bFGF;
epidermal growth factor (EGF); transforming growth factor (TGF)
such as TGF-alpha and TGF-beta, including TGF-b1, TGF-b2, TGF-b3,
TGF-b4, or TGF-b5; a tumor necrosis factor (TNF) such as TNF-alpha
or TNF-beta; insulin-like growth factor-I and -II (IGF-I and
IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor
binding proteins; CD proteins such as CD3, CD4, CD8, CD19, CD20,
CD22 and CD40; erythropoietin; osteoinductive factors;
immunotoxins; a bone morphogenetic protein (BMP); an interferon
such as interferon-alpha, -beta, and -gamma; colony stimulating
factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF; interleukins (ILs),
e.g., IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9 and
IL-10; superoxide dismutase; T-cell receptors; surface membrane
proteins; decay accelerating factor; viral antigen such as, for
example, a portion of the AIDS envelope; transport proteins; homing
receptors; addressins; regulatory proteins; integrins such as
CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and VCAM; a tumor
associated antigen such as HER2, HER3 or HER4 receptor; and
fragments of any of the above-listed polypeptides.
[0084] Exemplary molecular targets for antibodies encompassed by
the present invention include CD proteins such as CD3, CD4, CD8,
CD19, CD20, CD22, CD34 and CD40; members of the ErbB receptor
family such as the EGF receptor, HER2, HER3 or HER4 receptor; B
cell surface antigens, such as CD20 or BR3; a member of the tumor
necrosis receptor superfamily, including DRS; prostate stem cell
antigen (PSCA); cell adhesion molecules such as LFA-1, Mac1,
p150.95, VLA-4, ICAM-1, VCAM, alpha4/beta7 integrin, and
alpha4/beta3 integrin including either alpha or beta subunits
thereof (e.g. anti-CD11a, anti-CD18 or anti-CD11b antibodies);
growth factors such as VEGF as well as receptors therefor; tissue
factor (TF); a tumor necrosis factor (TNF) such as TNF-alpha or
TNF-beta, alpha interferon (alpha-IFN); an interleukin, such as
IL-8; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB)
receptor; mpl receptor; CTLA-4; protein C etc.
[0085] Soluble antigens or fragments thereof, optionally conjugated
to other molecules, can be used as immunogens for generating
antibodies. For transmembrane molecules, such as receptors,
fragments of these (e.g. the extracellular domain of a receptor)
can be used as the immunogen. Alternatively, cells expressing the
transmembrane molecule can be used as the immunogen. Such cells can
be derived from a natural source (e.g. cancer cell lines) or may be
cells which have been transformed by recombinant techniques to
express the transmembrane molecule. Other antigens and forms
thereof useful for preparing antibodies will be apparent to those
in the art.
[0086] Exemplary antibodies which can be formulated according to
the present invention include, but are not limited to the
following: anti-ErbB antibodies, including anti-HER2 antibodies
(e.g. trastuzumab or pertuzumab); antibodies that bind to a B-cell
surface marker, such as CD20 (for example rituximab and humanized
2H7/ocrelizumab), CD22, CD40 or BR3; antibodies that bind to IgE,
including omalizumab (XOLAIR.RTM.) commercially available from
Genentech, E26, HAE1, IgE antibody with an amino acid substitution
at position 265 of an Fc region thereof (US 2004/0191244 A1),
Hu-901, an IgE antibody as in WO2004/070011, or antibody that binds
the small extracellular segment on IgE, M1' (e.g. 47H4v5; see U.S.
Pat. No. 8,071,097), see, also, Presta et al., J. Immunol.
151:2623-2632 (1993); International Publication No. WO 95/19181;
U.S. Pat. No. 5,714,338, issued Feb. 3, 1998; U.S. Pat. No.
5,091,313, issued Feb. 25, 1992; WO 93/04173 published Mar. 4,
1993; WO 99/01556 published Jan. 14, 1999; and U.S. Pat. No.
5,714,338; antibodies that bind to vascular endothelial growth
factor (VEGF) (e.g. bevacizumab) or a VEGF receptor; anti-IL-8
antibodies (St John et al., Chest, 103:932 (1993), and
International Publication No. WO 95/23865); anti-PSCA antibodies
(WO01/40309); anti-CD40 antibodies, including S2C6 and humanized
variants thereof (WO00/75348); anti-CD11a antibodies, including
efalizumab (RAPTIVA.RTM.) (U.S. Pat. No. 5,622,700, WO 98/23761,
Steppe et al., Transplant Intl. 4:3-7 (1991), and Hourmant et al.,
Transplantation 58:377-380 (1994)); anti-CD18 antibodies (U.S. Pat.
No. 5,622,700, issued Apr. 22, 1997, or as in WO 97/26912,
published Jul. 31, 1997); anti-Apo-2 receptor antibody (WO 98/51793
published Nov. 19, 1998); anti-TNF-alpha antibodies including cA2
(REMICADE.RTM.) and adalimumab (HUMIRA.RTM.), CDP571 and MAK-195
(Afelimomab) (See, U.S. Pat. No. 5,672,347 issued Sep. 30, 1997,
Lorenz et al. J. Immunol. 156(4):1646-1653 (1996), and Dhainaut et
al. Crit. Care Med. 23(9):1461-1469 (1995)); anti-Tissue Factor
(TF) (European Patent No. 0 420 937 B1 granted Nov. 9, 1994);
anti-human .alpha.4.beta..sub.7 integrin (WO 98/06248 published
Feb. 19, 1998); anti-EGFR antibodies, including chimerized or
humanized 225 antibody as in WO 96/40210 published Dec. 19, 1996;
anti-CD3 antibodies, such as OKT3 (U.S. Pat. No. 4,515,893 issued
May 7, 1985); anti-CD25 or anti-tac antibodies such as CHI-621
(SIMULECT.RTM.) and (ZENAPAX.RTM.) (See U.S. Pat. No. 5,693,762
issued Dec. 2, 1997); anti-CD4 antibodies such as the cM-7412
antibody (Choy et al. Arthritis Rheum 39(1):52-56 (1996));
anti-CD52 antibodies such as alemtuzumab (CAMPATH-1H.RTM.)
(Riechmann et al. Nature 332:323-337 (1988); anti-Fc receptor
antibodies such as the M22 antibody directed against Fc.gamma.RI as
in Graziano et al. J. Immunol. 155(10):4996-5002 (1995);
anti-carcinoembryonic antigen (CEA) antibodies such as hMN-14
(Sharkey et al. Cancer Res. 55(23Suppl): 5935s-5945s (1995);
antibodies directed against breast epithelial cells including
huBrE-3, hu-Mc 3 and CHL6 (Ceriani et al. Cancer Res. 55(23):
5852s-5856s (1995); and Richman et al. Cancer Res. 55(23 Supp):
5916s-5920s (1995)); antibodies that bind to colon carcinoma cells
such as C242 (Litton et al. Eur J. Immunol. 26(1):1-9 (1996));
anti-CD38 antibodies, e.g. AT 13/5 (Ellis et al. J. Immunol.
155(2):925-937 (1995)); anti-CD33 antibodies such as Hu M195
(Jurcic et al. Cancer Res 55(23 Suppl):5908s-5910s (1995) and
CMA-676 or CDP771; anti-CD22 antibodies such as LL2 or LymphoCide
(Juweid et al. Cancer Res 55(23 Suppl):5899s-5907s (1995);
anti-EpCAM antibodies such as 17-1A (PANOREX.RTM.); anti-GpIIb/IIIa
antibodies such as abciximab or c7E3 Fab (REOPRO.RTM.); anti-RSV
antibodies such as MEDI-493 (SYNAGIS.RTM.); anti-CMV antibodies
such as PROTOVIR.RTM.; anti-HIV antibodies such as PRO542;
anti-hepatitis antibodies such as the anti-Hep B antibody
OSTAVIR.RTM.; anti-CA 125 antibody OvaRex; anti-idiotypic GD3
epitope antibody BEC2; anti-uvi33 antibody VITAXIN.RTM.; anti-human
renal cell carcinoma antibody such as ch-G250; ING-1; anti-human
17-1A antibody (3622W94); anti-human colorectal tumor antibody
(A33); anti-human melanoma antibody R24 directed against GD3
ganglioside; anti-human squamous-cell carcinoma (SF-25); and
anti-human leukocyte antigen (HLA) antibodies such as Smart ID10
and the anti-HLA DR antibody Oncolym (Lym-1); anti-CCRS(PRO 140);
ABT-325; ABT-308; ABT-147; anti-beta7 (etrolizumab); anti-HER3/EGFR
DAF (DL11f); anti-interleukin 6 receptor (IL6R) such as tocilizumab
(ACTEMRA.RTM.); and anti-Abeta (see WO2003/070760 and
WO2008/011348), etc.
[0087] In one embodiment the antibody which is formulated herein
binds CD20 and is selected from: rituximab, ocrelizumab/humanized
2H7 (Genentech), ofatumumab (WO 04/035607, Genmab, Denmark),
framework patched/humanized 1F5 (WO03/002607, Leung, S.), AME-133
(Applied Molecular Evolution), and humanized A20 antibody (US
2003/0219433, Immunomedics).
[0088] In one embodiment the antibody which is formulated binds
HER2 and is trastuzumab or pertuzumab.
[0089] In one embodiment the antibody which is formulated binds
VEGF and is bevacizumab.
[0090] In one embodiment the antibody that is formulated herein is
a humanized antibody.
[0091] In one embodiment the antibody that is formulated is a
recombinant antibody.
[0092] In one embodiment the antibody that is formulated has been
expressed by a recombinant Chinese Hamster Ovary (CHO) cell.
[0093] In one embodiment the antibody that is formulated is a full
length antibody.
[0094] In one embodiment the antibody that is formulated is a full
length human IgG1 antibody.
[0095] In one embodiment the antibody that is formulated is a full
length humanized IgG1 antibody.
[0096] In one embodiment the antibody that is formulated is a full
length recombinant humanized IgG1 antibody.
[0097] In one embodiment the antibody that is formulated is a full
length humanized IgG1 antibody that has been expressed by a
recombinant Chinese Hamster Ovary (CHO) cell.
[0098] In one embodiment the antibody that is formulated binds an
antigen selected from: CD20 (e.g. rituximab), HER2 (e.g.
trastuzumab), VEGF (bevacizumab), IL6R (tocilizumab), beta?
(etrolizumab), Abeta, HER3 and EGFR (DL11f), and M1' (47H4v5).
[0099] In one embodiment the antibody formulated is rituximab.
[0100] In one embodiment the antibody formulated is
trastuzumab.
[0101] In one embodiment the antibody formulated is
bevacizumab.
III. The Pre-Spray Dried Preparation
[0102] A preparation of the monoclonal antibody is generally
prepared which is to be subjected to spray drying, the so-called
"pre-spray dried preparation" herein.
[0103] In one embodiment, the pre-spray dried preparation comprises
a monoclonal antibody preparation which has been subjected to one
or more prior purification steps, such as affinity chromatography
(e.g. protein A chromatography), hydrophobic interaction
chromatography, ion exchange chromatography (anion and/or cation
exchange chromatography), virus filtration, etc. Thus, the antibody
preparation may be purified, essentially pure, and/or essentially
homogeneous.
[0104] In one embodiment, the monoclonal antibody in the pre-spray
dried preparation is concentrated. Exemplary methods for
concentrating the antibody include filtration (such as tangential
flow filtration or ultrafiltration), dialysis etc.
[0105] The pre-spray dried preparation may be liquid or frozen,
[0106] The pH of the pre-spray dried preparation is optionally
adjusted by a buffer. The buffer may for example have a pH from
about 4 to about 8, e.g. from about 5 to 7, for example 5.8 to 6.2,
and, in one embodiment, is approximately 6.0. A histidine buffer is
an exemplified embodiment herein. The concentration of the buffer
is dictated, at least in part, by the desired pH. Exemplary
concentrations for the buffer are from about 1 mM to about 200 mM,
or from about 10 mM to about 40 mM.
[0107] The pre-spray dried preparation optionally also comprises
one or more stabilizers which prevent denaturation and/or
aggregation of the antibody during the spray drying process.
Examples of such stabilizers include saccharides (e.g. sucrose or
trehalose) and/or surfactants (e.g. polysorbate 20 or polysorbate
80) and/or amino acids (e.g. histidine, arginine, glycine, and/or
alanine). The stabilizers are generally added in amount(s) which
protect and/or stabilize the monoclonal antibody at the lowest
amount of stabilizer possible, to avoid increasing the viscosity of
the final formulation.
[0108] With respect to saccharide stabilizers, such as
disaccharides (e.g. trehalose or sucrose), the molar ratio of
saccharide: monoclonal antibody (or disaccharide: monoclonal
antibody) is optionally from about 50 to about 400:1, e.g. from
about 100 to about 250:1. Stated differently, exemplary saccharide
concentrations in the pre-spray dried preparation are, for example,
from about 10 mM to about 1M, for example from about 50 mM to about
300 mM.
[0109] With respect to surfactant (if included in the pre-spray
drying formulation), polysorbate 20 or polysorbate 80 are examples
of surfactants that can be included. The surfactant is generally
included in an amount which reduces or prevents denaturation and/or
aggregation of the monoclonal antibody during the spray drying
process. The surfactant (e.g. polysorbate 20 or polysorbate 80)
concentration is optionally from about 0.0001% to about 1.0%, for
example from about 0.01% to about 0.1%.
[0110] The pre-spray dried preparation may be subjected to spray
drying procedures such as those described in the following
section.
IV. Spray Drying the Preparation
[0111] Spray drying herein is distinct from freeze drying commonly
used to prepare monoclonal antibody formulations insofar as it is
performed at temperatures above ambient temperature. Spray drying
temperatures are commonly expressed as "air inlet" and "air outlet"
temperatures. In one embodiment, the spray drying is performed at
an air inlet temperature from about 100.degree. C. to about
220.degree. C. (for example from about 120.degree. C. to about
160.degree. C.) and an air outlet temperature from about 50.degree.
C. to about 100.degree. C. (for example from about 60.degree. C. to
about 80.degree. C.).
[0112] The spray drying process generally comprises: atomization of
the liquid feed; drying of the droplets; and separation or recovery
of the dried product.
[0113] Embodiments of atomizers herein include: rotary atomizers,
pneumatic nozzle atomizers, ultrasonic nozzle atomizers, sonic
nozzles, etc.
[0114] The contact between the liquid feed and the drying air can
occur in two different modes. In a co-current system, drying air
and particles (droplets) move through the drying chamber in the
same direction. When drying air and droplets move in an opposite
direction, this is called a counter-current mode. Particles
produced in counter-current mode usually show a higher temperature
than the exhausting air. The exhausted air itself can leave the
system or can be recirculated. By choosing from the various spray
dryer designs (size, atomizer, aseptic conditions, etc.) and
adjusting the different process parameters (drying air flow, drying
air temperature, etc.), the final powder properties like particle
size, shape and structure or even sterility can be modified. If the
resulting moisture of the recovered powder is not sufficiently low,
post-treatment might be required, e.g., in the form of fluid bed
dryers and coolers, contact dryers or even microwave dryers.
[0115] When the liquid feed is atomized, its surface to mass ratio
is increased, the heat transfer between the air and the droplets is
accelerated, and droplets can dry relatively rapidly. Two
convection processes may be involved: heat transfer (air to
droplet) and mass transfer of moisture (droplet to air). In the
latter, moisture permeates through the boundary layer that
surrounds each droplet. Transfer rates may be influenced by
temperature, humidity, transport properties of the surrounding air,
droplet diameter and relative velocity between droplet and air.
[0116] The last step of a spray drying process is typically the
separation of the powder from the air/gas and the removal of the
dried product. In some embodiments, this step is as effective as
possible to obtain high powder yields and to prevent air pollution
through powder emission to the atmosphere. To this end, various
methods are available such as cyclones, bag filters, electrostatic
precipitators, high pressure gas, electrostatic charge and
combinations thereof.
[0117] The spray drying process produces particles comprising the
monoclonal antibody.
[0118] In one embodiment, the characteristics of the spray dried
powder comprise any one or more or the following:
[0119] (a) average particle size: from about 2 microns to about 30
microns; e.g. from about 2 microns to about 10 microns;
[0120] (b) particle morphology: predominantly spherical particles,
some dimples or holes in particles, "dry raisin" shape;
[0121] (c) water content: less than about 10%, for example less
than about 5%, e.g., where water content is measured by a chemical
titration method (e.g. Karl Fischer method) or a weight-loss method
(high-temperture heating); and
[0122] (d) stability: e.g., assessed by suspending the particles in
a vehicle and evaluating physical stability and/or chemical
stability and/or biological activity of the suspension preparation.
In one embodiment, the percentage monomer of such preparation is
95% to 100%, e.g. as evaluated by size exclusion chromatography
(SEC).
V. The Suspension Formulation
[0123] The spray dried monoclonal antibody particles prepared as
described in the preceding section are combined with a non-aqueous
suspension vehicle to generate the suspension formulation. This
formulation is suitable for administration to a subject. Generally,
the suspension formulation will not be subjected to either prior,
or subsequent, lyophilization or crystallization. In one
embodiment, a subcutaneous administration device (e.g. a pre-filled
syringe) is filled with the suspension formulation and used for
administering the formulation (see below for more detailed
disclosure regarding devices and methods of treatment).
[0124] The invention also provides a method of making a suspension
formulation comprising suspending the spray dried monoclonal
antibody in a non-aqueous suspension vehicle.
[0125] In one embodiment the antibody concentration in the
suspension formulation is about 200 mg/mL or more.
[0126] In one embodiment the antibody concentration in the
suspension formulation is from about 200 mg/mL to about 500
mg/mL.
[0127] In one embodiment the antibody concentration in the
suspension formulation is from about 250 mg/mL to about 400
mg/mL.
[0128] In one embodiment the antibody concentration in the
suspension formulation is from about 250 mg/mL to about 350
mg/mL.
[0129] The non-aqueous suspension vehicle preferably has a
viscosity at 25.degree. C., which is less than about 20 centipoise,
for example, less than about 10 centipoise, and optionally less
than than about 5 centipoise.
[0130] According to one embodiment of the invention, the viscosity
of the suspension formulation is from about 5 to about 100
centipoise, for instance, from about 10 to about 70 centipoise at
25.degree. C. In one embodiment, viscosity of the suspension
formulation is measured using a cone and plate rheometer (e.g. a
AR-G2 TA Instrument rheometer).
[0131] In one embodiment, the average particle size in the
suspension formulation is from about 2 microns to about 30 microns,
for example from about 5 microns to about 10 microns.
[0132] In one embodiment, the suspension formulation has an
injection glide force of less than about 20 newton, for example
less than about 15 newton. Such injection glide force may be
determined as a function of monoclonal antibody concentration by
injecting 1-mL suspension using a 1-mL long syringe through a
27-gauge TW staked needle in 10 seconds.
[0133] In one embodiment the non-aqueous suspension vehicle is
selected from: propylene glycol dicarprylate/dicaprate, benzyl
benzoate, ethyl lactate, or mixtures of two or three thereof,
[0134] In one embodiment the non-aqueous suspension vehicle
comprises ethyl lactate.
[0135] In one embodiment, the non-aqueous suspension vehicle
comprises a mixture of at least two non-aqueous
[0136] suspension vehicles: Vehicle A plus Vehicle B, wherein the
viscosity of Vehicle A is less than that of Vehicle B, but the
monoclonal antibody stability in Vehicle B is greater than that in
Vehicle A. An embodiment of such mixture is exemplified by the
mixture of ethyl lactate and propylene glycol
dicarprylate/dicaprate (for example).
[0137] In one aspect, the suspension formulation comprises a spray
dried full length human IgG1 monoclonal antibody at a concentration
from about 200 mg/mL to about 400 mg/mL suspended in a non-aqueous
suspension vehicle with a viscosity less than about 20 centipoise,
wherein the formulation has an average particle size from about 2
microns to about 10 microns, and injection glide force less than
about 15 newton.
[0138] The suspension formulation optionally further comprises one
or more excipients or stabilizers. Examples of such stabilizers
include saccharides (e.g. sucrose or trehalose) and/or surfactants
(e.g. polysorbate 20 or polysorbate 80) and/or amino acids (e.g.
histidine, arginine, glycine, and/or alanine). The stabilizers are
generally present in an amount which protects and/or stabilizes the
monoclonal antibody at the lowest amount of stabilizer possible, to
avoid increasing the viscosity of the suspension formulation. In
one embodiment, the stabilizers are present in the suspension
formulation as a result of having been added to the pre-spray dried
preparation, and/or have been added to the suspension formulation,
as desired.
[0139] With respect to saccharide stabilizers, such as
disaccharides (e.g. trehalose or sucrose), the molar ratio of
saccharide: monoclonal antibody (or disaccharide: monoclonal
antibody) in the suspension formulation is optionally from about 50
to about 400:1, e.g. from about 100 to about 250:1. Stated
differently, exemplary saccharide concentrations in the suspension
formulation are from about 10 mM to about 1 M, for example from
about 50 mM to about 300 mM.
[0140] With respect to surfactant (if included in the pre-spray
dried preparation), polysorbate 20 or polysorbate 80 are examples
of surfactants which can be present in the suspension formulation.
The surfactant (e.g. polysorbate 20 or polysorbate 80)
concentration is optionally from about 0.0001% to about 1.0%, for
example from about 0.01% to about 0.1%.
[0141] The suspension formulation is generally sterile, and this
can be achieved according to the procedures known to the skilled
person for generating sterile pharmaceutical formulations suitable
for administration to human subjects, including filtration through
sterile filtration membranes, prior to, or following, preparation
of the suspension formulation.
[0142] Moreover, the formulation is desirably one which has been
demonstrated to be stable upon storage. Various stability assays
are available to the skilled practitioner for confirming the
stability of the formulation. Stability can be tested by evaluating
physical stability, chemical stability, and/or biological activity
of the antibody in the suspension formulation around the time of
formulation as well as following storage at different temperatures
and time-points. In one embodiment, monoclonal antibody stability
is assessed by size distribution (percentage monomer, aggregation,
and/or fragmentation) before and after spray drying (e.g. before
and after spray drying over 3-month storage under the accelerated
temperature of 40.degree. C.). In one embodiment, size distribution
is assessed using size exclusion chromatography-high performance
liquid chromatography (SEC-HPLC). In one embodiment, the percentage
monomer loss in the suspension formulation (as measured by
SEC-HPLC) over 3 months is less than about 10%, for example less
than about 5%.
[0143] In one embodiment, the invention provides a method of making
a pharmaceutical formulation comprising preparing the suspension
formulation as described herein, and evaluating any one or more of
the following properties of the formulation:
[0144] (a) physical stability, chemical stability, and/or
biological activity of the monoclonal antibody in the suspension
(e.g. measuring percentage monomer using size exclusion
chromatography);
[0145] (b) viscosity of the suspension formulation;
[0146] (c) injectability or injection glide force of the suspension
formulation;
[0147] (d) surface energy analysis (SEA) or heat of sorption, e.g.
by inverse gas chromatography (IGC) to evaluate particle-suspension
vehicle interaction;
[0148] (e) particle size (e.g. average and/or peak particle size,
e.g. by laser diffraction analyzer); and/or
[0149] (e) suspension physical stability (settling, homogeneity
over time, particle sedimentation rate, etc).
[0150] Further detail of exemplary assays for these properties is
provided in the example below.
[0151] One or more additional other pharmaceutically acceptable
carriers, excipients or stabilizers such as those described in
Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed.
(1980) may be included in the formulation provided that they do not
adversely affect the desired characteristics of the formulation.
Acceptable carriers, excipients or stabilizers are nontoxic to
recipients at the dosages and concentrations employed and include;
additional buffering agents; co-solvents; antioxidants including
ascorbic acid and methionine; chelating agents such as EDTA; metal
complexes (e.g. Zn-protein complexes); biodegradable polymers such
as polyesters; preservatives; and/or salt-forming counterions such
as sodium.
VI. Medicaments and Treatments Using the Suspension Formulation
[0152] In one embodiment, the invention provides a method of
treating a disease or disorder in a subject comprising
administering the suspension formulation described herein to a
subject in an amount effective to treat the disease or
disorder.
[0153] Thus, the invention provides: the suspension formulation as
described herein for treating a patient in need of treatment with
the monoclonal antibody in the suspension formulation; and use of
the suspension formulation in the preparation of a medicament for
treating a patient in need of treatment with the monoclonal
antibody in the suspension formulation. In an alternative
embodiment, the invention provides: the formulation as described
herein for treating a disease or disorder in a patient; and use of
the formulation in the preparation of a medicament for treating a
disease or disorder in a patient.
[0154] In addition, the invention provides a method of treating a
patient comprising administering the formulation described herein
to a patient in order to treat a disease or disorder in the
subject. Preferably the formulation is administered subcutaneously
to the subject or patient. In one embodiment, the formulation is
administered by a pre-filled syringe containing the formulation
therein.
[0155] Where the antibody in the formulation binds to HER2, the
suspension formulation is preferably used to treat cancer. The
cancer will generally comprise HER2-expressing cells, such that the
HER2 antibody herein is able to bind to the cancer cells. Thus, the
invention in this embodiment concerns a method for treating
HER2-expressing cancer in a subject, comprising administering the
HER2 antibody pharmaceutical formulation to the subject in an
amount effective to treat the cancer. Exemplary cancers to be
treated herein with a HER2 antibody (e.g. trastuzumab or
pertuzumab) are HER2-positive breast cancer or gastric cancer.
[0156] Where the antibody in the formulation binds to a B-cell
surface marker such as CD.sub.2O, the formulation may be used to
treat a B-cell malignancy, such as NHL or CLL, or an autoimmune
disease (e.g. rheumatoid arthritis or vasculitis).
[0157] Where the antibody in the formulation binds VEGF (e.g.
bevacizumab), the formulation may be used to inhibit angiognesis,
treat cancer (such as colorectal, non-small cell lung (NSCL),
glioblastoma, breast cancer, and renal cell carcinoma), or treat
age-related macular degeneration (AMD) or macular edema.
[0158] Where the indication is cancer, the patient may be treated
with a combination of the suspension formulation, and a
chemotherapeutic agent. The combined administration includes
coadministration or concurrent administration, using separate
formulations or a single pharmaceutical formulation, and
consecutive administration in either order, wherein there is a time
period when both (or all) active agents simultaneously exert their
biological activities. Thus, the chemotherapeutic agent may be
administered prior to, or following, administration of the
composition. In this embodiment, the timing between at least one
administration of the chemotherapeutic agent and at least one
administration of the formulation is preferably approximately 1
month or less, and most preferably approximately 2 weeks or less.
Alternatively, the chemotherapeutic agent and the formulation are
administered concurrently to the patient, in a single formulation
or separate formulations.
[0159] Treatment with the suspension formulation will result in an
improvement in the signs or symptoms of the disease or disorder.
Moreover, treatment with the combination of the chemotherapeutic
agent and the antibody formulation may result in a synergistic, or
greater than additive, therapeutic benefit to the patient.
[0160] The formulation is administered to a human patient in accord
with known methods, such as intravenous administration, e.g., as a
bolus or by continuous infusion over a period of time, by
intramuscular, intraperitoneal, intracerobrospinal, subcutaneous,
intra-articular, intrasynovial, or intrathecal administration.
[0161] Intramuscular or subcutaneous administration of antibody
composition is preferred, with subcutaneous administration being
most preferred.
[0162] For subcutaneous delivery, the formulation may be
administered via syringe (e.g. pre-filled syringe); autoinjector;
injection device (e.g. the INJECT-EASE.TM. and GENJECT.TM. device);
injector pen (such as the GENPEN.TM.); or other device suitable for
administering a suspension formulation subutaneously. The preferred
device herein is a pre-filled syringe.
[0163] For the prevention or treatment of disease, the appropriate
dosage of the monoclonal antibody will depend on the type of
disease to be treated, as defined above, the severity and course of
the disease, whether the monoclonal antibody is administered for
preventive or therapeutic purposes, previous therapy, the patient's
clinical history and response to the monoclonal antibody, and the
discretion of the attending physician. The antibody is suitably
administered to the patient at one time or over a series of
treatments. Depending on the type and severity of the disease,
about 1 hg/kg to 50 mg/kg (e.g. 0.1-20 mg/kg) of antibody is an
initial candidate dosage for administration to the patient,
whether, for example, by one or more separate administrations, or
by continuous infusion. The dosage of the antibody will generally
be from about 0.05 mg/kg to about 10 mg/kg. If a chemotherapeutic
agent is administered, it is usually administered at dosages known
therefor, or optionally lowered due to combined action of the drugs
or negative side effects attributable to administration of the
chemotherapeutic agent. Preparation and dosing schedules for such
chemotherapeutic agents may be used according to manufacturers'
instructions or as determined empirically by the skilled
practitioner. Preparation and dosing schedules for such
chemotherapy are also described in Chemotherapy Service Ed., M. C.
Perry, Williams & Wilkins, Baltimore, Md. (1992).
VII. Articles of Manufacture
[0164] The invention herein also concerns a device with the
suspension formulation therein. Preferably the device is a
subcutaneous administration device, such as a pre-filled
syringe.
[0165] In a related aspect, the invention provides a method of
making an article of manufacture comprising filling a container
with the suspension formulation.
[0166] Embodiments of the container in the article of manufacture
include: syringes (such as pre-filled syringe), autoinjectors,
bottles, vials (e.g. dual chamber vials), and test tubes, etc. The
container holds the suspension formulation and the label on, or
associated with, the container may indicate directions for use. The
article of manufacture may further include other materials
desirable from a commercial and user standpoint, including other
buffers, diluents, filters, needles, syringes, and package inserts
with instructions for use as noted in the previous section.
[0167] The invention will be more fully understood by reference to
the following examples. They should not, however, be construed as
limiting the scope of the invention. All literature and patent
citations are incorporated herein by reference.
EXAMPLES
[0168] Developing high-concentration monoclonal antibody liquid
formulations (.gtoreq.200 mg/mL) for subcutaneous (SC)
administration is often challenging with increased viscosity that
makes injection difficult. This investigation was intended to
overcome this obstacle using a non-aqueous powder suspension
approach. Three human IgG1 monoclonal antibodies were spray dried
and suspended in a suspension vehicle at different monoclonal
antibody concentrations. Propylene glycol dicaprylate/dicaprate,
benzyl benzoate, and ethyl lactate were employed as model
suspension vehicles. Suspensions were characterized for viscosity,
particle size, and syringeability. Physical stability of the
suspension was visually inspected. The suspensions in general
outperformed the liquid solutions in terms of injectability despite
higher viscosity at the same monoclonal antibody concentrations.
Powder formulations and powder properties appeared to have little
effect on suspension viscosity or injectability. Among the three
suspension vehicles, ethyl lactate suspensions had the lowest
viscosity, below 20 centipoise, and lowest syringe injection glide
force, below 15 newton, at monoclonal antibody concentration as
high as 333 mg/mL (total powder concentration at 500 mg/mL).
Inverse gas chromatography (IGC) analysis of the suspension
supported the conclusion that the suspension vehicle was the most
important factor impacting suspension performance. Ethyl lactate
rendered greater heat of sorption than other suspension vehicles.
Without being bound by any one theory, this indicates that strong
particle-suspension vehicle interaction may reduce
particle-particle self association, leading to low suspension
viscosity and glide force. Ethyl lactate suspensions, however,
lacked the physical suspension stability exhibited by propylene
glycol dicaprylate/dicaprate and benzyl benzoate. Specific mixtures
of ethyl lactate and propylene glycol dicaprylate/dicaprate
improved the overall suspension performance in high monoclonal
antibody concentration suspensions.
[0169] Amongst other things, these examples demonstrated the
viability of high monoclonal antibody concentration (>300 mg/mL)
in suspension formulations for SC administration.
MATERIALS AND METHODS
[0170] Three recombinant chimeric/humanized monoclonal antibodies
of the human IgG1 subclass bevacizumab, trastuzumab and rituximab
were manufactured by Genentech (South San Francisco, Calif.). These
antibodies were expressed by Chinese hamster ovary (CHO) cell
lines. All antibody drug substance liquid solutions were
concentrated to 100 mg/mL using a tangential-flow filtration unit
(PELLICON3.RTM. 10 kD, Millipore, Billerica, Mass.) and formulated
with trehalose dihydrate. All bulks were buffered to a pH of
.about.6.0. For antibody powder suspension preparation, propylene
glycol dicaprylate/dicaprate (Batch #091125, SASOL, Hamburg,
Germany), benzyl benzoate (Cat # B9550, Sigma-Aldrich, St Loius,
Mo.), and ethyl lactate (Lot #BCBC7752, Sigma-Aldrich, St. Louis,
Mo.) were used as suspension vehicles.
Spray Drying
[0171] Two types of spray dryers were used in this study, a
pilot-scale unit (MS-35, SPX Flow Technology Systems, Inc.,
Elkridge, Md.) and a bench-top unit (B-191, Buchi Corp., New
Castle, Del.). MS-35 is approximately 2-fold larger capacity than
B-191, i.e., 2.5 vs. 1.6 kg/hour of the maximum water evaporation
rate and 35 vs. 20 kg/hour maximum compressed air consumption rate.
The pilot-scale unit was constructed mostly of stainless steel with
heat insulation (drying chamber, cyclone, etc.) while the bench-top
unit was made of glass. The pilot scale unit was equipped with a
high-efficiency cyclone. To calculate the yield of powder
collection, only the powder collected in the receiver was
considered for the pilot-scale unit, and the powder collected on
the cyclone and the receiver lid was included for the bench-top
unit. The spray drying conditions and the characteristics dry
powders produced using both spray dryers are listed in Table 2.
TABLE-US-00002 TABLE 2 Spray-drying conditions in two types of
spray dryers and characterization results of three antibodies
formulated with trehalose at 1:2 antibody:trehalose weight ratio
Monoclonal Antibody Type Bevacizumab Trastuzumab Rituximab Drying
Spray Dryer Pilot Bench-top Pilot Bench-top Pilot Bench-top
Condition Inlet Temp. 182 134 182 138 182 136 (.degree. C.) Outlet
Temp. 87 88 87 89 87 88 (.degree. C.) Liq Feed Rate 12 3 12 3 13 3
(mL/min) Liq Vol Dried 250 50 250 50 250 50 (mL) Yield (%) 99 60
100 65 98 59 Particle Size (D.sub.50) (.mu.m) 9.6 2.5 8.8 2.8 10.6
5.1 Water Content (%) 4.0 7.6 4.7 6.9 5.0 8.8
Freeze Drying
[0172] Monoclonal antibody solutions were also freeze-dried to
compare the dry-state stability with spray dried samples. Liquid
formulations were aliquoted in 1 mL into 2 cc glass vials placed
with butyl stoppers, then placed on pre-chilled shelves at
-50.degree. C. in a lyophilizer (Model# LYOMAX2.RTM., BOC Edward,
Tewksbury, Mass.). The samples were dried by lowering the pressure
to 100 mTorr and increasing the shelf temperature to -25.degree. C.
during the primary drying, followed by the secondary drying at
35.degree. C. The total lyophylization cycle time was approximately
60 hours.
Particle Size Analysis
[0173] The particle size distribution was measured using a laser
diffraction analyzer (LA-950, Horiba Instruments, Kyoto, Japan).
The LA-950 consists of two light sources (blue LAD, red laser), a
sample handling system to control the interaction of particles and
incident light, and an array of high quality photodiodes to detect
the scattered light over a wide range of angles. The scattered
light collected on the detectors was used to calculate the particle
size distribution of the sample analyzed using the Mie Theory. For
spray dried samples, several milligrams of the dry powders were
dispersed in 50 mL of isopropyl alcohol in the MiniFlow cell
attached on LA-950 and sonicated using the sonicator also attached
on LA-950 for about one minutes prior to analysis. For particles
suspended in vehicles were diluted with each vehicle in
FractionCell and mix with a stirrer attached on LA-950 prior to
analysis.
Density Analysis
[0174] The density of the powder was determined by mixing 500 mg of
powder in 4 mL of propylene glycol dicaprylate/dicaprate oil in a
volumetric cylinder and measuring the displaced oil volume as the
powder volume. Powder density can be calculated using powder weight
and volume.
Scanning Electron Microscopy
[0175] Surface morphology of spray dried samples was examined using
an environmental scanning electron microscope (XL30, FEL,
Hillsboro, Oreg.). Each sample was mounted on aluminum stubs and
sputter coated with 10 nm layer of AuPd, and scanned at a voltage
of 2 kV, and the photographs were taken at magnifications of 1000
and 2000.
Water Content Analysis
[0176] Residual moisture in spray dried samples were determined
using volumetric Karl Fischer titration analyzer (DL31,
Mettler-Toledo). Approximately 100 mg of each sample was injected
into the titration cell that contained anhydrous methanol. Hydranal
composite 2 volumetric reagent (Cat#34696, Hiedel-deHaen,
Heidelberg, Germany) was used as a titrant.
Size Exclusion Chromatography
[0177] The quantitation of size variants was determined by size
exclusion chromatography. This analysis utilized a G3000SW).sub.XL
column, 7.8 mm ID.times.30 cm, 5 .mu.m (TOSOH BioScience) run on an
HPLC system (1100, Agilent). The mobile phases are 0.2 M potassium
phosphate and 0.25 M potassium chloride at pH 6.2 for bevacizumab,
0.1 M potassium phosphate at pH 6.8 for trastuzumab, and 0.2 M
potassium phosphate and 0.25 M potassium chloride at pH 7.0 for
rituximab. The chromatography was run isocractically at a flow rate
of 0.5 mL/min for 30 minutes. The column temperature was maintained
at ambient for bevacizumab and rituximab, and 30.degree. C. for
trastuzumab, and the eluent absorbance was monitored at 280 nm.
Each monoclonal antibody was diluted with its respective
formulation buffer to 25 mg/mL for bevacizumab and 10 mg/mL for
both trastuzumab and rituximab. Their injection volume is 10 .mu.L
for bevacizumab and for 20 .mu.L for both trastuzumab and
rituximab.
Monoclonal Antibody Physical Stability in Spray Dried and
Freeze-Dried Powder Formulations
[0178] Spray dried and freeze-dried powder samples were aliquotted
into 2 cc glass vial, approximately 25 monoclonal antibody. Each
vial was sealed with a rubber stopper and FLIP-OFF.RTM. cap and
stored at 40.degree. C. for up to 3 months. At the stability time
points of time zero (immediately after drying), 1, 2, 3 months,
each dry sample was reconstituted with 1 mL of purified water, and
the antibody physical stability was determined by protein size
distribution (% monomer, aggregation, and fragmentation) using
SEC-HPLC.
Preparation of Suspension Formulations
[0179] The powder was weighed onto a 2-mL vial. Based on the powder
density determined, the appropriate amount of suspension vehicle
was added to prepare the powder concentration in the unit of mg of
powder in 1 mL of suspension volume. Samples were then homogenized
for 2 minutes at 7500 rpm using a 0.5-cm tip probe on a Tempest
Virtishear homogenizer (Virits Corp, Gardiner, N.Y.).
Viscosity Measurement
[0180] The viscosity of solution and suspension samples was
measured using a cone and plate rheometer (AR-G2 TA Instrument, New
Castle, Del.). Each sample was loaded onto the lower measuring
plate and allowed to come to thermal equilibrium at 25.degree. C. A
solvent trap equipped on AR-G2 was used to prevent solution
evaporation during the measurement. The sample viscosity was
measured every 10 seconds for 2 minutes using a cone with a 20 mm
diameter and 1 degree angle at shear rate of 1000 per second.
Syringe Glide Force Measurement
[0181] One mL of suspension was drawn into a 1.0 mL-Long 27G TW
1/2'' staked needle syringe (BD, Franklin Lakes, N.J.) sealed with
a plunger stopper (W4023/FLT, West Pharmaceutical, Lionville, Pa.).
The internal barrel of the syringe was coated with 0.5 mg silicone
oil (Dow 360 Medical Fluid, 1,000 cSt). A Material Testing System
(Model 5542, Instron, Grove City, Pa.) with a load cell was used to
apply a steady compression rate of 190 mm/min. The gliding force
profile was analyzed and established as a function of the distance
of the plunger rod travelling inside the syringe barrel.
Inverse Gas Chromatography (IGC)
[0182] IGC experiment was performed using a Surface Energy Analysis
(SEA) System (MSM-iGC 2000, Surface Measurement Services Ltd,
Allentown, Pa.). Approximately 200 mg of powder sample was packed
into individual silanised glass columns and both ends of columns
were sealed using silanised glass wool to prevent sample movement.
The specific surface areas of the powder samples were determined by
measuring the Octane adsorption isotherms at 30.degree. C. and 0%
RH from the IGC SEA. The BET specific surface areas of the samples
were subsequently calculated from their corresponding octane
isotherms, within the partial pressure range (10% to 35%
P/P.sub.0). Decane, nonane, octane and heptane were used as alkane
probes for dispersive surface energy determination. Specific
acid-based Gibbs free energy was also measured using acetone,
acetonitrile, ethanol and ethyl acetate. For heat of sorption
measurement, the suspension vehicles were used as the gaseous
probes. All samples were pre-conditioned in-situ with a carrier gas
of helium at 30.degree. C. for 2 hours, and all the measurements
were conducted at 30.degree. C. with a carrier gas flow rate of 10
cm.sup.3/sec.
RESULTS AND DISCUSSION
Spray Dried Antibody/Trehalose Powders
[0183] Three types of monoclonal antibodies were formulated in
liquid solutions containing trehalose, serving as a carbohydrate
stabilizer to monoclonal antibody, at the weight ratio of 1:2 of
trehalose:antibody prior to spray drying. This low weight ratio is
equivalent to approximately 220:1 molar ratio was used for the
purpose of minimizing its volume contribution, which was below the
minimum molar ratio of 300:1 commonly used for sugar to stabilize
proteins as a lyoprotectant (Shire et al., J Pharm Sci 93:1390-1402
(2004)). Note that a 400 mg powder/mL suspension represents a 270
mg antibody/mL concentration even at the 1:2 weight ratio of
trehalose:antibody, which was at the low limit of the target
antibody concentration for this study.
[0184] Three monoclonal antibodies formulated at 100 mg/mL with 50
mg/mL trehalose, were spray dried using a bench-top spray dryer
(B-191) and a pilot-scale spray dryer (MS-35). Spray-drying
conditions and powder characterization results are summarized in
Table 2. Comparable outlet temperatures of 87-89.degree. C. were
employed for all samples because outlet temperature was considered
the key parameter dictating the spray-drying capability (Maa et
al., Pharm. Dev Technol 2:213-223 (1997); Lee G. Spray Drying of
Proteins, in "Rational Protein Formulation: Theory and Practice"
(Eds. Carpenter J, Manning M), Pharmaceutical Biotechnology Series
(Ed. Borchardt R). Plenum Press, pp. 135-158 (2002); Maury et al.
Eur. J. Pharm. Biopharm. 59:566-573 (2005); Maa et al. Biotech.
Bioeng. 60:301-309 (1998); and Maa et al. J. Pharm. Sci. 87:152-159
(1998)). The pilot-scale spray dryer demonstrated better
performance in powder collection yield (>96%) and water content
of 4-5%, while the samples dried by the bench-top spray dryer had
60% yield and 7-9% water content. The pilot-scale dryer was also
capable of producing larger particles of 8-11 .mu.m (D.sub.50)
whereas the bench-top dryer produced 2-5 .mu.m (D.sub.50)
particles. The advantages of the pilot-scale dryer can be
attributed to efficient energy use and greater powder collection
efficiency. Particle shape and morphology for all antibodies was
generally spherical with dimples, which were antibody dependent.
The type of the spray dryer did not affect particle morphology.
Overall, dryer performance and the antibody type resulted in some
degree of variations in particle properties. Although these
variations are not dramatic, they allowed us to evaluate their
effect on suspension performance.
Antibody Physical Stability in Spray dried and Freeze-Dried Powder
Formulations
[0185] A general concern about spray drying of biologics was high
temperature stress, particularly for the pilot dryer which had
higher inlet temperature of >180.degree. C. Antibody physical
stability of the dry samples was determined upon reconstitution
with purified water by protein size distribution (% monomer,
aggregation and fragmentation) using SEC-HPLC before and after
spray drying over 3-month storage under the accelerated temperature
of 40.degree. C. (FIG. 1). Despite the high drying temperature used
in the pilot-scale spray dryer, the impact of the drying process on
(%) monomer was minimal. The antibody physical stability for spray
dried bevacizumab and trastuzumab was compared to the freeze-dried
counterparts by monitoring the change in (%) monomer at 40.degree.
C. over 3 months. The (%) monomer for all samples decreased at the
accelerated condition mainly due to aggregation, which is not
surprising given the sub-optimal amount of trehalose to protect
antibody in the formulation. However, the spray dried samples had
greater antibody physical stability than the freeze-dried samples.
The (%) monomer of spray dried trastuzumab and bevacizumab
decreased by .about.2% and .about.4% respectively, whereas both
freeze-dried antibodies suffered a greater (%) monomer loss of
.about.6.5% over 3 months, despite their lower water content of
.about.0.8%. Thus, spray drying is a viable approach, from the
process and stability perspective, in making antibody powders for
suspension formulation development.
Selection of Suspension Vehicles
[0186] The primary criterion for the selection of the suspension
vehicle was low viscosity, preferably <10 Cp, as suspension
vehicle viscosity would contribute to suspension viscosity in a
linear fashion based on Einstein's Equation for the viscosity of
solutions (Einstein, A., Annalen der Physik 34:591-92 (1911)).
.eta.=.eta..sub.o(1+2.5.phi.) (Equation 2)
Where .eta. is the suspension viscosity, .eta..sub.o the viscosity
of pure suspension vehicle, and .phi. the volume fraction of the
solute.
[0187] The three suspension vehicles selected for this study,
propylene glycol dicaprylate/dicaprate, benzyl benzoate, and ethyl
lactate, met this criterion (Table 3). MIGLYOL 840.RTM. is
propylene glycol diesters of caprylic and capric acids from the
MIGLYOL.RTM. neutral oil family. MIGLYOL 810.RTM. and MIGLYOL
812.RTM. have been approved for intravenous and intramuscular
injections but they are viscous, >30 cp at ambient temperature.
Propylene glycol dicaprylate/dicaprate, the least viscous in the
family (.about.9 cp), has been used for transdermal applications
(Mahjour et al., Intl J Phann 95:161-169 (1999); Seniro, W., Intl J
Toxicol 18:35-52 (1999)). Benzyl benzoate is similar to propylene
glycol dicaprylate/dicaprate in viscosity, .about.9 cp, and has
often been used as a preservative in liquid injectables at <10%
concentration. Ethyl lactate has been used commonly in
pharmaceutical preparations, food additives, and fragrances due to
its relatively low toxicity. Although ethyl lactate has not yet
been parenterally approved, it had low toxicity in mice for
intramuscular and intravenous injection (Spiegel and Noseworthy, J
Pharm Sci 52:917-927 (1963); Mottu et al., PDA J. Pharm. Sci.
Technol. 54:456-469 (2000)). Ethyl lactate has a water-like
viscosity, .about.2 cp.
TABLE-US-00003 TABLE 3 Structure, viscosity, and pharmaceutical
application information of three model suspension vehicles tested
in this study Miglyol 840 Ethyl Lactate Structure ##STR00001##
##STR00002## Viscosity (cp) at 20.degree. C. 9 2 Pharmaceutical Not
currently approved for Used as flavor enhancer for Applications
parenteral use, but some animal oral dose medications. Not tox
studies have been approved for parenteral use conducted for skin
delivery but acute toxcity in mice by SC and IV are available
Benzyl Benzoate Stucture ##STR00003## Viscosity (cp) at 20.degree.
C. 9 Pharmaceutical Used as a preservative in Applications liquid
dosage form for parenteral administration in quantities less than
10%
Effect of Antibody Type and Powder Properties on Suspension
Viscosity
[0188] All antibodies dried by both bench-top and pilot-scale spray
dryers (Table 2) were suspended in propylene glycol
dicaprylate/dicaprate. Suspension viscosity was measured as a
function of antibody concentration, and compared to the antibody
liquid solutions (FIG. 2). Suspension viscosity for all antibodies
was similar in the range of antibody concentration tested,
suggesting that variations in antibody types and powder properties
(particle size, morphology, and moisture content) had little effect
on suspension viscosity. Suspension viscosity increased with
increasing antibody concentration in an exponential manner, which
can be expressed as:
.eta..sub.Miglyol 840=8.24e.sup.0.0088(powder cone) (Equation
3)
Certainly, it is very different from the Einstein equation
(Equation 2) which is primarily for dilute suspensions. Equation 4,
a modified version of Equation 2, took the interactions of more
concentrated suspensions into consideration (Kunitz, M., J. General
Physiology pages 715-725 (July 1926)), however, it still
significantly underestimated the empirical data (see the dash line
in FIG. 2).
.eta./.eta..sub.o=(1+0.5.phi.)/(1-.phi.).sup.4 (Equation 4)
[0189] It was interesting to find that suspension viscosity was
actually higher than the viscosity of the corresponding antibody
liquid solution at the same antibody concentration. No difference
in suspension viscosity was observed among the antibodies, although
the type of antibody did significantly affect liquid viscosity.
Surface Energies of Spray dried Powders by IGC
[0190] Kanai and co-workers (Kanai et al., J. Pharm. Sci.
97:4219-4227 (2005)) found reversible self-association as the
result of Fab-Fab interactions in their viscosity study tested with
two antibodies made of the same construct with different amino acid
sequences in the complementarity determining region (CDR) region in
aqueous solutions. Such viscosity differences due to the antibody
types in powder suspensions in non-aqueous vehicles were not
observed (FIG. 2). This observation could be interpreted from the
perspective of particle surface energy distribution in the powder
suspension. Particle surface energy, the combination of polar and
non-polar (dispersive) energy components, can dictate the level of
interactions with suspension vehicles and particles. IGC is a
common tool for surface energy measurement. The particle's
dispersive surface energy using decane, nonane, octane and heptane
as the probes, and also specific acid-base (polar) Gibbs free
energy were measured using acetone, ethyl acetate, ethanol, and
acetonitrile as the probes. Surface energy is a distribution in
response to particle size distribution of the powder sample but
only surface energies at the 50% values were reported in Table 4.
The dispersive surface energy, .gamma..sub.50, was in a narrow
range of 36 to 38 mJ/m.sup.2 for all three antibodies. The
differences in specific acid-base Gibbs free energy,
.DELTA.G.sub.50, of these antibodies in response to the four
acid-base probes were also in a narrow range of 8 to 13 mJ/m.sup.2.
The comparable surface energy distribution among the three antibody
powders could explain similar particle-suspension vehicle and
particle-particle interactions, leading to their comparable
suspension viscosity in propylene glycol dicaprylate/dicaprate
(FIG. 2).
TABLE-US-00004 TABLE 4 Dispersive surface energy (.gamma..sub.50),
specific acid-base Gibbs free energy (.DELTA.G.sub.50), and heat of
sorption of spray dried monoclonal antibody powders (all measured
using IGC) Heat of Sorption .DELTA.H.sub.sorption Powder Suspension
Vehicles .gamma..sub.50 (mJ/m.sup.2) .DELTA.G.sub.50 (mJ//m.sup.2)
(KJ/mole) Bevacizumab Decane, nonane, octane and 37.5 heptane
Trastuzumab Decane, nonane, octane and 36.8 heptane Rituximab
Decane, nonane, octane and 38.3 heptane Bevacizumab Acetone 8.4
Ethyl acetate 6.2 Ethanol 14.8 Acetonitrile 12.9 Trastuzumab
Acetone 8.2 Ethyl acetate 6.6 Ethanol 14.5 Acetonitrile 12.7
Rituximab Acetone 8.4 Ethyl acetate 7.3 Ethanol 14.9 Acetonitrile
12.8 Bevacizumab Propylene glycol 39.9 .+-. 0.5
dicaprylate/dicaprate Benzyl benzoate 36.5 .+-. 0.7 Ethyl lactate
51.5 .+-. 0.3 Rituximab Propylene glycol 43.4 .+-. 0.5
dicaprylate/dicaprate Benzyl benzoate 42.8 .+-. 0.6 Ethyl lactate
58.5 .+-. 0.4
Injectability of Suspensions in Three Vehicles
[0191] Injectability can be monitored by glide force measurement,
which is a performance indicator more relevant than viscosity
measurement. The glide force of the rituximab powder suspension in
three vehicles was determined as a function of antibody
concentration by injecting 1-mL suspension using a 1-mL long
syringe through a 27-gauge TW staked needle in 10 seconds (FIG. 3).
The glide force for all suspensions increased with antibody
concentration, however, it was below 20 N even at 200 mg/mL
antibody concentration despite the high viscosity (FIG. 2). The
predicted glide force for the antibody liquid solutions extracted
from FIG. 4 in Reference 3 was higher than the suspension glide
force. The glide force in ethyl lactate suspension was lowest among
the three suspension vehicles tested. The glide force of the ethyl
lactate suspension at 333 mg antibody/mL was equivalent to that in
the other two suspension vehicles at about half of the antibody
concentration (167 mg/mL), which was still below the target
threshold of 15 newton, even at high antibody concentration of 333
mg/mL. The reasons for the viscosity-glide force relationship
discrepancy between the liquid solution and the suspension are not
clear.
Effect of Suspension Vehicle on Suspension Viscosity
[0192] Suspension viscosity was tested in three vehicles containing
the spray dried rituximab powder (FIG. 4). The viscosity in ethyl
lactate was the lowest among the three vehicles; the viscosity of
the ethyl lactate suspension at 333 mg antibody/mL was equivalent
to that of the suspension in propylene glycol dicaprylate/dicaprate
and benzyl benzoate at about half of the antibody concentration
(167 mg/mL).
Heat of Sorption by IGC and Particle Size
[0193] Heat of sorption (.DELTA.H.sub.sorption) is a direct measure
of the strength of the interactions between a solid and gas
molecules adsorbed on the surface (Thielmann F., "Inverse gas
chromatography: Characterization of alumina and related surfaces,"
In "Encyclopedia of Surface and Colloid Science Volume 4 (edit by
P. Somasundaran) CRC Press, Boca Raton, Fla., p 3009-3031 (2006);
Thielmann and Butler, "Heat of sorption on microcrystalline
cellulose by pulse inverse gas chromatography at infinite
dilution," Surface Measurement Services Application Note 203
(http://www.thesorptionsolution.com/Information_Application_Notes_IGC.php-
#Aps) (2007)).
[0194] The IGC method was employed to measure the heat of sorption
between spray dried particles and the suspension vehicles (Table
4). For both bevacizumab and rituximab, ethyl lactate suspension
had higher heat of sorption than the other two suspension vehicles.
Particle size of the suspension particles was also compared among
the three suspensions (FIG. 5). The peak particle size (highest
percentage) was 28, 25, and 7 mm for propylene glycol
dicaprylate/dicaprate, benzyl benzoate and ethyl lactate,
respectively. Both heat of sorption and particle size data show
that the higher heat of sorption in ethyl lactate suspensions
indicated higher particle-suspension vehicle interaction than
particle-particle interaction and that the degree of particle
self-association in ethyl lactate was lower than that in propylene
glycol dicaprylate/dicaprate or benzyl benzoate.
Suspension Physical Stability
[0195] Despite low viscosity and glide force in the ethyl lactate
suspension, it displayed a peculiar suspension physical stability
as a function of time. The powder in the ethyl lactate suspension
settled to the bottom and floated to the surface of the suspension
after 1-day ambient storage (FIG. 6A). Homogeneity of the ethyl
lactate suspension could be restored by vortexing (FIG. 6B). On the
contrary, the suspension physical stability in propylene glycol
dicaprylate/dicaprate was much more stable and remained well
suspended over two weeks (FIG. 6C).
[0196] According to the particle sedimentation rate determined by
Stoke's Law (Eq. 4 below), the particles in ethyl lactate would
settle approximately 4.5 times faster than in propylene glycol
dicaprylate/dicaprate, based on the density and viscosity of ethyl
lactate and propylene glycol dicaprylate/dicaprate, 1.03 g/cm.sup.3
and 0.92 g/cm.sup.3, and 2 cP and 9 cP, respectively. Thus, Stoke's
Law alone couldn't fully explain the observation of extremely fast
settlement of particles in ethyl lactate as compared to propylene
glycol dicaprylate/dicaprate, suggesting other mechanisms such as
surface electrical charge (i.e., zeta potential) may play a role.
However, the phenomenon of some of the particles floating to the
top of ethyl lactate surface is difficult to explain because the
density of the spray dried particles is higher than ethyl
lactate.
s=d(.rho..sub.s-.rho..sub.1)g/(18.eta.) (Equation 5)
where s is sedimentation rate, d diameter of the particle,
.rho..sub.s the density of the particle, .rho..sub.1 the density of
the suspension vehicle, g acceleration due to gravity, and .eta.
the viscosity of the suspension vehicle.
Suspension Vehicle Mixture to Improve Suspension Performance
[0197] The mixtures of ethyl lactate and propylene glycol
dicaprylate/dicaprate were used as suspension vehicles for testing
rituximab suspension physical stability. Particle size was
determined for these mixture suspensions (FIG. 7A). The particle
size decreased with decreasing propylene glycol
dicaprylate/dicaprate contribution in the mixture where the peak
particle size was 28, 13, 11, 8 and 7 .mu.m for propylene glycol
dicaprylate/dicaprate:ethyl lactate mixture at 100:0, 75:25, 50:50,
25:75, and 0:100, respectively. From the suspension physical
stability perspective, the poor suspension stability of ethyl
lactate was improved by mixing with a small amount of propylene
glycol dicaprylate/dicaprate as demonstrated in FIG. 7B where
homogeneous suspension was maintained for rituximab powder in 25:75
propylene glycol dicaprylate/dicaprate:ethyl lactate mixture after
2-week ambient storage. It was demonstrated that overall suspension
performance can be improved using a suspension vehicle mixture.
CONCLUSIONS
[0198] These examples demonstrated that the non-aqueous powder
suspension approach was feasible for high monoclonal antibody
concentration SC administration. Dry powder preparation by
spray-drying was scalable using the high efficiency spray-drying
process. The most important parameter for overall suspension
performance was determined to be the type of suspension vehicle.
Powder suspension in ethyl lactate displayed excellent suspension
injectability with a low glide force of <15 N via a 27-gauge TW
staked needle for antibody concentration as high as 333 mg/mL
(total powder concentration of 500 mg/mL). Without being bound by
any one theory, low viscosity and injectability could be attributed
to strong particle-suspension vehicle interaction that prevents
particle-particle agglomeration into larger particle size in the
suspension. However, this mechanism did not support physical
suspension stability. Dry antibody particles had a higher tendency
to settle out in the ethyl lactate suspension than in propylene
glycol dicaprylate/dicaprate. The approach of using suspension
vehicle mixture proved to be effective in improving overall
suspension performance.
Sequence CWU 1
1
301451PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 1Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu
Val Lys Pro Gly Ala 1 5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser
Gly Tyr Thr Phe Thr Ser Tyr 20 25 30 Asn Met His Trp Val Lys Gln
Thr Pro Gly Arg Gly Leu Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro
Gly Asn Gly Asp Thr Ser Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys
Ala Thr Leu Thr Ala Asp Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met
Gln Leu Ser Ser Leu Thr Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Ser Thr Tyr Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly
100 105 110 Ala Gly Thr Thr Val Thr Val Ser Ala Ala Ser Thr Lys Gly
Pro Ser 115 120 125 Val Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser
Gly Gly Thr Ala 130 135 140 Ala Leu Gly Cys Leu Val Lys Asp Tyr Phe
Pro Glu Pro Val Thr Val 145 150 155 160 Ser Trp Asn Ser Gly Ala Leu
Thr Ser Gly Val His Thr Phe Pro Ala 165 170 175 Val Leu Gln Ser Ser
Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val 180 185 190 Pro Ser Ser
Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His 195 200 205 Lys
Pro Ser Asn Thr Lys Val Asp Lys Lys Ala Glu Pro Lys Ser Cys 210 215
220 Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly
225 230 235 240 Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp
Thr Leu Met 245 250 255 Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val
Val Asp Val Ser His 260 265 270 Glu Asp Pro Glu Val Lys Phe Asn Trp
Tyr Val Asp Gly Val Glu Val 275 280 285 His Asn Ala Lys Thr Lys Pro
Arg Glu Glu Gln Tyr Asn Ser Thr Tyr 290 295 300 Arg Val Val Ser Val
Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly 305 310 315 320 Lys Glu
Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile 325 330 335
Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val 340
345 350 Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val
Ser 355 360 365 Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile
Ala Val Glu 370 375 380 Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr
Lys Thr Thr Pro Pro 385 390 395 400 Val Leu Asp Ser Asp Gly Ser Phe
Phe Leu Tyr Ser Lys Leu Thr Val 405 410 415 Asp Lys Ser Arg Trp Gln
Gln Gly Asn Val Phe Ser Cys Ser Val Met 420 425 430 His Glu Ala Leu
His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser 435 440 445 Pro Gly
Lys 450 2213PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 2Gln Ile Val Leu Ser Gln Ser Pro Ala
Ile Leu Ser Ala Ser Pro Gly 1 5 10 15 Glu Lys Val Thr Met Thr Cys
Arg Ala Ser Ser Ser Val Ser Tyr Ile 20 25 30 His Trp Phe Gln Gln
Lys Pro Gly Ser Ser Pro Lys Pro Trp Ile Tyr 35 40 45 Ala Thr Ser
Asn Leu Ala Ser Gly Val Pro Val Arg Phe Ser Gly Ser 50 55 60 Gly
Ser Gly Thr Ser Tyr Ser Leu Thr Ile Ser Arg Val Glu Ala Glu 65 70
75 80 Asp Ala Ala Thr Tyr Tyr Cys Gln Gln Trp Thr Ser Asn Pro Pro
Thr 85 90 95 Phe Gly Gly Gly Thr Lys Leu Glu Ile Lys Arg Thr Val
Ala Ala Pro 100 105 110 Ser Val Phe Ile Phe Pro Pro Ser Asp Glu Gln
Leu Lys Ser Gly Thr 115 120 125 Ala Ser Val Val Cys Leu Leu Asn Asn
Phe Tyr Pro Arg Glu Ala Lys 130 135 140 Val Gln Trp Lys Val Asp Asn
Ala Leu Gln Ser Gly Asn Ser Gln Glu 145 150 155 160 Ser Val Thr Glu
Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser Ser 165 170 175 Thr Leu
Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr Ala 180 185 190
Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser Phe 195
200 205 Asn Arg Gly Glu Cys 210 3121PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
3Gln Val Gln Leu Gln Gln Pro Gly Ala Glu Leu Val Lys Pro Gly Ala 1
5 10 15 Ser Val Lys Met Ser Cys Lys Ala Ser Gly Tyr Thr Phe Thr Ser
Tyr 20 25 30 Asn Met His Trp Val Lys Gln Thr Pro Gly Arg Gly Leu
Glu Trp Ile 35 40 45 Gly Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser
Tyr Asn Gln Lys Phe 50 55 60 Lys Gly Lys Ala Thr Leu Thr Ala Asp
Lys Ser Ser Ser Thr Ala Tyr 65 70 75 80 Met Gln Leu Ser Ser Leu Thr
Ser Glu Asp Ser Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Thr Tyr
Tyr Gly Gly Asp Trp Tyr Phe Asn Val Trp Gly 100 105 110 Ala Gly Thr
Thr Val Thr Val Ser Ala 115 120 4106PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
4Gln Ile Val Leu Ser Gln Ser Pro Ala Ile Leu Ser Ala Ser Pro Gly 1
5 10 15 Glu Lys Val Thr Met Thr Cys Arg Ala Ser Ser Ser Val Ser Tyr
Ile 20 25 30 His Trp Phe Gln Gln Lys Pro Gly Ser Ser Pro Lys Pro
Trp Ile Tyr 35 40 45 Ala Thr Ser Asn Leu Ala Ser Gly Val Pro Val
Arg Phe Ser Gly Ser 50 55 60 Gly Ser Gly Thr Ser Tyr Ser Leu Thr
Ile Ser Arg Val Glu Ala Glu 65 70 75 80 Asp Ala Ala Thr Tyr Tyr Cys
Gln Gln Trp Thr Ser Asn Pro Pro Thr 85 90 95 Phe Gly Gly Gly Thr
Lys Leu Glu Ile Lys 100 105 55PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 5Ser Tyr Asn Met His 1 5
617PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 6Ala Ile Tyr Pro Gly Asn Gly Asp Thr Ser Tyr Asn
Gln Lys Phe Lys 1 5 10 15 Gly 712PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 7Ser Thr Tyr Tyr Gly Gly
Asp Trp Tyr Phe Asn Val 1 5 10 810PRTArtificial SequenceDescription
of Artificial Sequence Synthetic peptide 8Arg Ala Ser Ser Ser Val
Ser Tyr Ile His 1 5 10 97PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 9Ala Thr Ser Asn Leu Ala Ser
1 5 109PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 10Gln Gln Trp Thr Ser Asn Pro Pro Thr 1 5
11453PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 11Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Tyr Thr Phe Thr Asn Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr
Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg
Phe Thr Phe Ser Leu Asp Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Lys Tyr Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val
100 105 110 Trp Gly Gln Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr
Lys Gly 115 120 125 Pro Ser Val Phe Pro Leu Ala Pro Ser Ser Lys Ser
Thr Ser Gly Gly 130 135 140 Thr Ala Ala Leu Gly Cys Leu Val Lys Asp
Tyr Phe Pro Glu Pro Val 145 150 155 160 Thr Val Ser Trp Asn Ser Gly
Ala Leu Thr Ser Gly Val His Thr Phe 165 170 175 Pro Ala Val Leu Gln
Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val 180 185 190 Thr Val Pro
Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val 195 200 205 Asn
His Lys Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys 210 215
220 Ser Cys Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu
225 230 235 240 Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys Asp Thr 245 250 255 Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val Asp Val 260 265 270 Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp Gly Val 275 280 285 Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr Asn Ser 290 295 300 Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp Trp Leu 305 310 315 320 Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala 325 330 335
Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro 340
345 350 Gln Val Tyr Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys Asn
Gln 355 360 365 Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp Ile Ala 370 375 380 Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys Thr Thr 385 390 395 400 Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser Lys Leu 405 410 415 Thr Val Asp Lys Ser Arg
Trp Gln Gln Gly Asn Val Phe Ser Cys Ser 420 425 430 Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser 435 440 445 Leu Ser
Pro Gly Lys 450 12214PRTArtificial SequenceDescription of
Artificial Sequence Synthetic polypeptide 12Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Val Leu Ile 35 40 45
Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Ser Thr
Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
Arg Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser
Asp Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu
Leu Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys
Val Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser
Val Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175
Ser Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180
185 190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys
Ser 195 200 205 Phe Asn Arg Gly Glu Cys 210 13123PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
13Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Tyr Thr Phe Thr Asn
Tyr 20 25 30 Gly Met Asn Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Gly Trp Ile Asn Thr Tyr Thr Gly Glu Pro Thr
Tyr Ala Ala Asp Phe 50 55 60 Lys Arg Arg Phe Thr Phe Ser Leu Asp
Thr Ser Lys Ser Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Tyr Pro His
Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp Val 100 105 110 Trp Gly Gln
Gly Thr Leu Val Thr Val Ser Ser 115 120 14108PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
14Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Ser Ala Ser Gln Asp Ile Ser Asn
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Val Leu Ile 35 40 45 Tyr Phe Thr Ser Ser Leu His Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Ser Thr Val Pro Trp 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Val Glu Ile Lys Arg 100 105 1510PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 15Gly
Tyr Thr Phe Thr Asn Tyr Gly Met Asn 1 5 10 1617PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 16Trp
Ile Asn Thr Tyr Thr Gly Glu Pro Thr Tyr Ala Ala Asp Phe Lys 1 5 10
15 Arg 1713PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 17Pro His Tyr Tyr Gly Ser Ser His Trp Tyr Phe Asp
Val 1 5 10 1811PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 18Ser Ala Ser Gln Asp Ile Ser Asn Tyr
Leu Asn 1 5 10 197PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 19Phe Thr Ser Ser Leu His Ser 1 5
209PRTArtificial SequenceDescription of Artificial Sequence
Synthetic peptide 20Gln Gln Tyr Ser Thr Val Pro Trp Thr 1 5
21449PRTArtificial SequenceDescription of Artificial Sequence
Synthetic polypeptide 21Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Asn Ile Lys Asp Thr 20 25 30 Tyr Ile His Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro
Thr Asn Gly Tyr Thr Arg Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Ala Asp Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ser Arg Trp Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln
100 105 110 Gly Thr Leu Val Thr Val Ser Ser Ala Ser Thr Lys Gly Pro
Ser Val 115 120 125 Phe Pro Leu Ala Pro Ser Ser Lys Ser Thr Ser Gly
Gly Thr Ala Ala 130 135 140 Leu Gly Cys Leu Val Lys
Asp Tyr Phe Pro Glu Pro Val Thr Val Ser 145 150 155 160 Trp Asn Ser
Gly Ala Leu Thr Ser Gly Val His Thr Phe Pro Ala Val 165 170 175 Leu
Gln Ser Ser Gly Leu Tyr Ser Leu Ser Ser Val Val Thr Val Pro 180 185
190 Ser Ser Ser Leu Gly Thr Gln Thr Tyr Ile Cys Asn Val Asn His Lys
195 200 205 Pro Ser Asn Thr Lys Val Asp Lys Lys Val Glu Pro Lys Ser
Cys Asp 210 215 220 Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro Glu
Leu Leu Gly Gly 225 230 235 240 Pro Ser Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile 245 250 255 Ser Arg Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu 260 265 270 Asp Pro Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His 275 280 285 Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg 290 295 300 Val
Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys 305 310
315 320 Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu 325 330 335 Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr 340 345 350 Thr Leu Pro Pro Ser Arg Glu Glu Met Thr Lys
Asn Gln Val Ser Leu 355 360 365 Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp Ile Ala Val Glu Trp 370 375 380 Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys Thr Thr Pro Pro Val 385 390 395 400 Leu Asp Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp 405 410 415 Lys Ser
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His 420 425 430
Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro 435
440 445 Gly 22214PRTArtificial SequenceDescription of Artificial
Sequence Synthetic polypeptide 22Asp Ile Gln Met Thr Gln Ser Pro
Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Ser
Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr Thr Thr
Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys Arg
Thr Val Ala Ala 100 105 110 Pro Ser Val Phe Ile Phe Pro Pro Ser Asp
Glu Gln Leu Lys Ser Gly 115 120 125 Thr Ala Ser Val Val Cys Leu Leu
Asn Asn Phe Tyr Pro Arg Glu Ala 130 135 140 Lys Val Gln Trp Lys Val
Asp Asn Ala Leu Gln Ser Gly Asn Ser Gln 145 150 155 160 Glu Ser Val
Thr Glu Gln Asp Ser Lys Asp Ser Thr Tyr Ser Leu Ser 165 170 175 Ser
Thr Leu Thr Leu Ser Lys Ala Asp Tyr Glu Lys His Lys Val Tyr 180 185
190 Ala Cys Glu Val Thr His Gln Gly Leu Ser Ser Pro Val Thr Lys Ser
195 200 205 Phe Asn Arg Gly Glu Cys 210 23120PRTArtificial
SequenceDescription of Artificial Sequence Synthetic polypeptide
23Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Asn Ile Lys Asp
Thr 20 25 30 Tyr Ile His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Arg Ile Tyr Pro Thr Asn Gly Tyr Thr Arg
Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Ala Asp
Thr Ser Lys Asn Thr Ala Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ser Arg Trp Gly Gly
Asp Gly Phe Tyr Ala Met Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu
Val Thr Val Ser Ser 115 120 24108PRTArtificial SequenceDescription
of Artificial Sequence Synthetic polypeptide 24Asp Ile Gln Met Thr
Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val
Thr Ile Thr Cys Arg Ala Ser Gln Asp Val Asn Thr Ala 20 25 30 Val
Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40
45 Tyr Ser Ala Ser Phe Leu Tyr Ser Gly Val Pro Ser Arg Phe Ser Gly
50 55 60 Ser Arg Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu
Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln His Tyr
Thr Thr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile
Lys Arg 100 105 2510PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 25Gly Phe Asn Ile Lys Asp Thr Tyr Ile
His 1 5 10 2617PRTArtificial SequenceDescription of Artificial
Sequence Synthetic peptide 26Arg Ile Tyr Pro Thr Asn Gly Tyr Thr
Arg Tyr Ala Asp Ser Val Lys 1 5 10 15 Gly 2711PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 27Trp
Gly Gly Asp Gly Phe Tyr Ala Met Asp Tyr 1 5 10 2811PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 28Arg
Ala Ser Gln Asp Val Asn Thr Ala Val Ala 1 5 10 297PRTArtificial
SequenceDescription of Artificial Sequence Synthetic peptide 29Ser
Ala Ser Phe Leu Tyr Ser 1 5 309PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 30Gln Gln His Tyr Thr Thr Pro
Pro Thr 1 5
* * * * *
References