U.S. patent application number 11/917188 was filed with the patent office on 2008-12-18 for self-buffering protein formulations.
Invention is credited to David Brems, Yatin R. Gokarn, Susan Irene Hershenson, Eva Kras, Richard Louis Remmele.
Application Number | 20080311078 11/917188 |
Document ID | / |
Family ID | 37570999 |
Filed Date | 2008-12-18 |
United States Patent
Application |
20080311078 |
Kind Code |
A1 |
Gokarn; Yatin R. ; et
al. |
December 18, 2008 |
Self-Buffering Protein Formulations
Abstract
The invention herein described, provides, among other things,
self-buffering protein formulations. Particularly, the invention
provides self-buffering pharmaceutical protein formulations that
are suitable for veterinary and human medical use. The
self-buffering protein formulations are substantially free of other
buffering agents, stably maintain pH for the extended time periods
involved in the distribution and storage of pharmaceutical proteins
for veterinary and human medical use. The invention further
provides methods for designing, making, and using the formulation.
In addition to other advantages, the formulations avoid the
disadvantages associated with the buffering agents conventionally
used in current formulations of proteins for pharmaceutical use.
The invention in these and other respects can be productively
applied to a wide variety of proteins and is particularly useful
for making and using self-buffering formulations of pharmaceutical
proteins for veterinary and medical use, especially, in particular,
for the treatment of diseases in human subjects.
Inventors: |
Gokarn; Yatin R.; (Seattle,
WA) ; Kras; Eva; (Seattle, WA) ; Remmele;
Richard Louis; (Camarillo, CA) ; Brems; David;
(Newbury Park, CA) ; Hershenson; Susan Irene;
(Newbury Park, CA) |
Correspondence
Address: |
LARRY S. MILLSTEIN;HOLLAND & KNIGHT LLP
1600 TYSONS BOULEVARD, SUITE 700
MCLEAN
VA
22102
US
|
Family ID: |
37570999 |
Appl. No.: |
11/917188 |
Filed: |
June 8, 2006 |
PCT Filed: |
June 8, 2006 |
PCT NO: |
PCT/US2006/022599 |
371 Date: |
June 16, 2008 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60690582 |
Jun 14, 2005 |
|
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|
Current U.S.
Class: |
514/1.1 ;
424/130.1; 424/172.1; 424/85.6; 424/85.7; 514/8.4; 514/8.6 |
Current CPC
Class: |
A61K 47/10 20130101;
C07K 2317/21 20130101; C07K 16/2851 20130101; C07K 16/2875
20130101; C07K 16/2803 20130101; A61K 39/3955 20130101; A61K 47/26
20130101; C07K 16/2866 20130101; C07K 2317/565 20130101; A61P 43/00
20180101; A61K 9/08 20130101; C07K 16/2827 20130101; A61K 47/02
20130101; C07K 16/00 20130101; A61K 39/39591 20130101; C07K 2317/94
20130101; C07K 16/241 20130101; A61P 35/00 20180101 |
Class at
Publication: |
424/85.5 ; 514/2;
424/130.1; 424/172.1; 424/85.6; 424/85.7 |
International
Class: |
A61K 38/21 20060101
A61K038/21; A61K 38/00 20060101 A61K038/00; A61P 43/00 20060101
A61P043/00; A61K 39/395 20060101 A61K039/395 |
Claims
1. A composition comprising a pharmaceutical protein, wherein at
the pH of the composition, 21.degree. C., one atmosphere, and
equilibrium with ambient atmosphere, the protein has a buffer
capacity per unit volume of at least that of approximately 4.0 mM
sodium acetate buffer in pure water in the range of pH 5.0 to 4.0
or pH 5.0 to 5.5 under the same conditions, wherein further,
exclusive of the buffer capacity of said protein, the buffer
capacity per unit volume of the composition under the same
conditions is no more than that of 2.0 mM sodium acetate buffer in
pure water in the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 under the
same conditions, wherein the composition has been approved for
pharmaceutical use by an authority legally empowered to grant such
approval.
2. A composition comprising a pharmaceutical protein, wherein at
the pH of the composition, 21.degree. C., one atmosphere, and
equilibrium with ambient atmosphere, the protein has a buffer
capacity per unit volume of at least 1.50 mEq/liter-pH unit,
wherein further, exclusive thereof, the buffer capacity per unit
volume of the composition is less than 0.5 mEq/liter-pH unit,
wherein the composition has been approved for pharmaceutical use by
an authority legally empowered to grant such approval.
3. A composition according to claim 1, wherein the protein provides
at least 80% of the buffer capacity of the composition.
4. A composition according to claim 3, wherein the concentration of
the protein is between approximately 20 and 400 mg/ml.
5. A composition according to claim 4, wherein the pH maintained by
the buffering action of the protein is between approximately 3.5
and 8.0.
6. A composition according to claim 5, wherein the pH maintained by
the buffering action of the protein is between approximately 4 and
6.
7. A composition according to claim 5, further comprising one or
more pharmaceutically acceptable salts, wherein the total salt
concentration is less than 150 mM.
8. A composition according to claim 7, further comprising one or
more pharmaceutically acceptable salts, wherein the total salt
concentration is less than 100 mM.
9. A composition according to claim 5, further comprising one or
more pharmaceutically acceptable polyols.
10. A composition according to claim 9, wherein the polyol is one
or more of sorbitol, mannitol, sucrose, trehalose, or glycerol.
11. A composition according to claim 5, further comprising one or
more pharmaceutically acceptable surfactants.
12. A composition according to claim 11, wherein the surfactant is
one or more of polysorbate 20, polysorbate 80, other fatty acid
esters of sorbitan, polyethoxylates, and poloxamer 188.
13. A composition according to claim 9, further comprising one or
more pharmaceutically acceptable surfactants.
14. A composition according to claim 1, further comprising one or
more pharmaceutically acceptable: osmotic balancing agents;
anti-oxidants; antibiotics; antimycotics; bulking agents;
lyoprotectants; anti-foaming agents; chelating agents;
preservatives; colorants; analgesics; or additional pharmaceutical
agents.
15. A composition according to claim 5, further comprising one or
more pharmaceutically acceptable: osmotic balancing agents;
anti-oxidants; antibiotics; antimycotics; bulking agents;
lyoprotectants; anti-foaming agents; chelating agents;
preservatives; colorants; analgesics; or additional pharmaceutical
agents.
16. A composition according to claim 7, further comprising one or
more pharmaceutically acceptable: osmotic balancing agents;
anti-oxidants; antibiotics; antimycotics; bulking agents;
lyoprotectants; anti-foaming agents; chelating agents;
preservatives; colorants; analgesics; or additional pharmaceutical
agents.
17. A composition according to claim 1, wherein the protein is or
comprises: an antibody, Fab fragment, Fab.sub.2 fragment, Fab.sub.3
fragment, Fc fragment, scFv fragment, bis-scFv(s) fragment,
minibody, diabody, triabody tetrabody, VhH domain, V-NAR domain,
V.sub.H domain, V.sub.L domain, camel Ig, Ig NAR, receptibody,
peptibody, or a variant or a derivative thereof or a protein
related thereto, or a modification thereof.
18. A composition according to claim 17, wherein the protein
comprises an Fc fragment or a part thereof, or a variant or a
derivative of an Fc fragment or a part thereof or a protein related
to an Fc fragment or part thereof, or a modification of any
thereof.
19. A composition according to claim 18, wherein the protein
further comprises a first binding moiety of a pair of cognate
binding moieties.
20. A composition according to claim 1, wherein the protein is
selected from the group consisting of proteins that bind
specifically to one or more CD proteins, HER receptor family
proteins, cell adhesion molecules, growth factors, nerve growth
factors, fibroblast growth factors, transforming growth factors
(TGF), insulin-like growth factors, osteoinductive factors,
insulins and insulin-related proteins, coagulation and
coagulation-related proteins, colony stimulating factors (CSFs),
other blood and serum proteins blood group antigens; receptors,
receptor-associated proteins, growth hormone receptors, T-cell
receptors; neurotrophic factors. neurotrophins, relaxins,
interferons, interleukins, viral antigens, lipoproteins, integrins,
rheumatoid factors, immunotoxins, surface membrane proteins,
transport proteins, homing receptors, addressins, regulatory
proteins, and immunoadhesins.
21. A composition according to claim 1, wherein the protein is
selected from the group consisting of: OPGL specific binding
proteins, myostatin specific binding proteins, IL-4 receptor
specific binding proteins, IL1-R1 specific binding proteins, Ang2
specific binding proteins, NGF-specific binding proteins, CD22
specific binding proteins, IGF-1 receptor specific binding
proteins, B7RP-1 specific binding proteins, IFN gamma specific
binding proteins, TALL-1 specific binding proteins, stem cell
factors, Flt-3 ligands, and IL-17 receptors.
22. A composition according to claim 1, wherein the protein is
selected from the group consisting of proteins that bind
specifically to one or more of: CD3, CD4, CD8, CD19, CD20, CD34;
HER2, HER3, HER4, the EGF receptor; LFA-1, Mol, p150,95, VLA-4,
ICAM-1, VCAM, alpha v/beta 3 integrin; vascular endothelial growth
factor ("VEGF"); growth hormone, thyroid stimulating hormone,
follicle stimulating hormone, luteinizing hormone, growth hormone
releasing factor, parathyroid hormone, mullerian-inhibiting
substance, human macrophage inflammatory protein (MIP-1-alpha),
erythropoietin (EPO), NGF-beta, platelet-derived growth factor
(PDGF), aFGF, bFGF, epidermal growth factor (EGF), TGF-alpha,
TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, IGF-I,
IGF-II, des(1-3)-IGF-I (brain IGF-I), insulin, insulin A-chain,
insulin B-chain, proinsulin, insulin-like growth factor binding
proteins;, such as, among others, factor VIII, tissue factor, von
Willebrands factor, protein C, alpha-1-antitrypsin, plasminogen
activators, such as urokinase and tissue plasminogen activator
("t-PA"), bombazine, thrombin, and thrombopoietin; M-CSF, GM-CSF,
G-CSF, albumin, IgE, flk2/flt3 receptor, obesity (OB) receptor,
bone-derived neurotrophic factor (BDNF), NT-3, NT-4, NT-5, NT-6);
relaxin A-chain, relaxin B-chain, prorelaxin; interferon-alpha,
-beta, and -gamma; IL-1 to IL-10; AIDS envelope viral antigen;
calcitonin, glucagon, atrial natriuretic factor, lung surfactant,
tumor necrosis factor-alpha and -beta, enkephalinase, RANTES, mouse
gonadotropin-associated peptide, Dnase, inhibin, and activin;
protein A or D, bone morphogenetic protein (BMP), superoxide
dismutase, decay accelerating factor (DAF).
23. A composition according to claim 1, wherein the protein is
selected from the group consisting of: Actimmune
(Interferon-gamma-1b), Activase (Alteplase), Aldurazme
(Laronidase), Amevive (Alefacept), Avonex (Interferon beta-1a),
BeneFIX (Nonacog alfa), Beromun (Tasonermin), Beatseron
(Interferon-beta-1b), BEXXAR (Tositumomab), Tev-Tropin
(Somatropin), Bioclate or RECOMBINATE (Recombinant), CEREZME
(Imiglucerase), ENBREL (Etanercept), Eprex (epoetin alpha),
EPOGEN/Procit (Epoetin alfa), FABRAZYME (Agalsidase beta),
Fasturtec/Elitek ELITEK (Rasburicase), FORTEO (Teriparatide),
GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon,
rDNA origin), GONAL-F (follitropin alfa), KOGENATE FS (Octocog
alfa), HERCEPTIN (Trastuzumab), HUMATROPE (SOMATROPIN), HUMIRA
(Adalimumab), Insulin in Solution, INFERGEN.RTM. (Interferon
alfacon-1), KINERET.RTM. (anakinra), Kogenate FS (Antihemophilic
Factor), LEUKIN (SARGRAMOSTIM Recombinant human
granulocyte-macrophage colony stimulating factor (rhuGM-CSF)),
CAMPATH (Alemtuzumab), RITUXAN.RTM. (Rituximab), TNKase
(Tenecteplase), MYLOTARG (gemtuzumab ozogamicin), NATRECOR
(nesiritide), ARANESP (darbepoetin alfa), NEULASTA (pegfilgrastim),
NEUMEGA (oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES
(Somatropin), NOVOSEVEN (Eptacog alfa), NUTROPIN AQ (somatropin),
Oncaspar (pegaspargase), ONTAK (denileukin diftitox), ORTHOCLONE
OKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS
(peginterferon alfa-2a), PROLEUKIN (Aldesleukin), PULMOZYME (domase
alfa), Retavase (Reteplase), REBETRON Combination Therapy
containing REBETOL.RTM. (Ribavirin) and INTRON.RTM. A (Interferon
alfa-2b), REBIF (interferon beta-1a), REFACTO (Antihemophilic
Factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO
(abciximab)ROFERON.RTM.-A (Interferon alfa-2a), SIMULECT
(baasiliximab), SOMAVERT (Pegivisomant), SYNAGIS.RTM.
(palivizumab), Stemben (Ancestim, Stem cell factor), THYROGEN,
INTRON.RTM. A (Interferon alfa-2b), PEG-INTRON.RTM. (Peginterferon
alfa-2b), XIGRIS.RTM. (Drotrecogin alfa activated), XOLAIR.RTM.
(Omalizumab), ZENAPAX.RTM. (daclizumab), and ZEVALIN.RTM.
(Ibritumomab Tiuxetan).
24. A composition according to claim 1, wherein the protein is
Ab-hOPGL or a fragment thereof, or a variant or derivative of
Ab-hOPGL or of a fragment thereof, or an Ab-hOPGL related protein
or fragment thereof, or a modification of any thereof.
25. A composition according to claim 1, wherein the protein is
Ab-hOPGL.
26. A composition according to claim 1, wherein the protein is
Ab-hIL4R or a fragment thereof, or a variant or derivative of
Ab-hIL4R or of a fragment thereof, or an Ab-hIL4R related protein
or fragment thereof, or a modification of any thereof.
27. A composition according to claim 1, wherein the protein is
Ab-hIL4R.
28. A composition according to claim 1, wherein the protein is
Ab-hB7RP1 or a fragment thereof, or a variant or derivative of
Ab-hB7RP1 or of a fragment thereof, or an Ab-hB7RP1 related protein
or fragment thereof, or a modification of any thereof.
29. A composition according to claim 1, wherein the protein is
Ab-hB7RP1.
30. A lyophilate which upon reconstitution provides a composition
according to claim 1.
31. A kit comprising in one or more containers a composition
according to claim 1, and instructions regarding the use
thereof.
32. A kit comprising in one or more containers a lyophilate
according to claim 31, and instructions regarding the use
thereof.
33. A method for treating a subject, comprising administering to a
subject in an amount and by a route effective for treatment, a
composition according to claim 1.
34. A process for preparing a composition according to claim 1,
comprising removing residual buffer using a counter ion.
35. A process for preparing a composition according to claim 34,
comprising removing residual buffer using any one or more of the
following in the presence of a counter ion: size exclusion
chromatography, dialysis, and/or tangential flow filtration.
36. A process for preparing a composition according to claim 35,
comprising removing residual buffer using ion exchange
chromatography.
37. A process for preparing a composition according to claim 1,
comprising removing residual buffer by diafiltration against a
bufferless solution having a pH below the desired pH.
38. A process for preparing a composition according to claim 37,
wherein following diafiltration the pH is adjusted to a desired pH
by addition of dilute acid and/or dilute base.
39. A lyophilate which upon reconstitution provides a composition
according to claim 20.
40. A kit comprising in one or more containers a composition
according to claim 20 and instructions regarding the use
thereof.
41. A kit comprising in one or more containers a lyophilate
according to claim 40, and instructions regarding the use
thereof.
42. A method for treating a subject, comprising administering to a
subject in an amount and by a route effective for treatment, a
composition according to claim 20.
43. A process for preparing a composition according to claim 20,
comprising removing residual buffer using a counter ion.
44. A process for preparing a composition according to claim 43,
comprising removing residual buffer using any one or more of the
following in the presence of a counter ion: size exclusion
chromatography, dialysis, and/or tangential flow filtration.
45. A process for preparing a composition according to claim 43,
comprising removing residual buffer using ion exchange
chromatography.
46. A process for preparing a composition according to claim 20,
comprising removing residual buffer by diafiltration against a
bufferless solution having a pH below the desired pH.
47. A process for preparing a composition according to claim 46,
wherein following diafiltration the pH is adjusted to a desired pH
by addition of dilute acid and/or dilute base.
48. A lyophilate which upon reconstitution provides a composition
according to claim 21.
49. A kit comprising in one or more containers a composition
according claim 21 and instructions regarding the use thereof.
50. A kit comprising in one or more containers a lyophilate
according to claim 49, and instructions regarding the use
thereof.
51. A method for treating a subject, comprising administering to a
subject in an amount and by a route effective for treatment, a
composition according to claim 21.
52. A process for preparing a composition according to claim 21,
comprising removing residual buffer using a counter ion.
53. A process for preparing a composition according to claim 52,
comprising removing residual buffer using any one or more of the
following in the presence of a counter ion: size exclusion
chromatography, dialysis, and/or tangential flow filtration.
54. A process for preparing a composition according to claim 52,
comprising removing residual buffer using ion exchange
chromatography.
55. A process for preparing a composition according to claim 21,
comprising removing residual buffer by diafiltration against a
bufferless solution having a pH below the desired pH.
56. A process for preparing a composition according to claim 55,
wherein following diafiltration the pH is adjusted to a desired pH
by addition of dilute acid and/or dilute base.
57. A lyophilate which upon reconstitution provides a composition
according to claim 23.
58. A kit comprising in one or more containers a composition
according claim 23 and instructions regarding the use thereof.
59. A kit comprising in one or more containers a lyophilate
according to claim 57, and instructions regarding the use
thereof.
60. A method for treating a subject, comprising administering to a
subject in an amount and by a route effective for treatment, a
composition according to claim 23.
61. A process for preparing a composition according to claim 23,
comprising removing residual buffer using a counter ion.
62. A process for preparing a composition according to claim 61,
comprising removing residual buffer using any one or more of the
following in the presence of a counter ion: size exclusion
chromatography, dialysis, and/or tangential flow filtration.
63. A process for preparing a composition according to claim 61,
comprising removing residual buffer using ion exchange
chromatography.
64. A process for preparing a composition according to claim 21,
comprising removing residual buffer by diafiltration against a
bufferless solution having a pH below the desired pH.
65. A process for preparing a composition according to claim 62,
wherein following diafiltration the pH is adjusted to a desired pH
by addition of dilute acid and/or dilute base.
Description
REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation-in-part of and claims
full priority benefit of U.S. Provisional Application Ser. No.
60/690,582 filed 14 Jun. 2005, which is incorporated herein by
reference in its entirety.
FIELD OF THE INVENTION
[0002] The invention relates to the formulation of proteins,
especially pharmaceutical proteins. In particular, it relates to
self-buffering biopharmaceutical protein compositions, and to
methods for designing, making, and using the compositions. It
further relates to pharmaceutical protein compositions for
veterinary and/or for human medical use, and to methods relating
thereto.
BACKGROUND OF THE INVENTION
[0003] Many aspects of pharmaceutical production and formulation
processes are pH sensitive. Maintaining the correct pH of a
finished pharmaceutical product is critical to its stability,
effectiveness, and shelf life, and pH is an important consideration
in designing formulations for administration that will be
acceptable, as well as safe and effective.
[0004] To maintain pH, pharmaceutical processes and formulations
use one or more buffering agents. A variety of buffering agents are
available for pharmaceutical use. The buffer or buffers for a given
application must be effective at the desired pH. They must also
provide sufficient buffer capacity to maintain the desired pH for
as long as necessary. A good buffer for a pharmaceutical
composition must satisfy numerous other requirements as well. It
must be appropriately soluble. It must not form deleterious
complexes with metal ions, be toxic, or unduly penetrate,
solubilize, or absorb on membranes or other surfaces. It should not
interact with other components of the composition in any manner
which decreases their availability or effectiveness. It must be
stable and effective at maintaining pH over the range of conditions
to which it will be exposed during formulation and during storage
of the product. It must not be deleteriously affected by oxidation
or other reactions occurring in its environment, such as those that
occur in the processing of the composition in which it is providing
the buffering action. If carried over or incorporated into a final
product, a buffering agent must be safe for administration,
compatible with other components of the composition over the
shelf-life of the product, and acceptable for administration to the
end user.
[0005] Although there are many buffers in general use, only a
limited number are suitable for biological applications and, of
these, fewer still are acceptable for pharmaceutical processes and
formulations. As a result, it often is challenging to find a buffer
that not only will be effective at maintaining pH but also will
meet all the other requirements for a given pharmaceutical process,
formulation, or product.
[0006] The challenge of finding a suitable buffer for
pharmaceutical use can be especially acute for pharmaceutical
proteins. The conformation and activity of proteins are critically
dependent upon pH. Proteins are susceptible to a variety of pH
sensitive reactions that are deleterious to their efficacy,
typically many more than affect small molecule drugs. For instance,
to mention just a few salient examples, the side chain amides of
asparagine and glutamine are deamidated at low pH (less than 4.0)
and also at neutral or high pH (greater than 6.0). Aspartic acid
residues promote the hydrolysis of adjacent peptide bonds at low
pH. The stability and disposition of disulfide bonds is highly
dependent on pH, particularly in the presence of thiols.
Solubility, flocculation, aggregation, precipitation, and
fibrillation of proteins are critically dependent on pH. The
crystal habit important to some pharmaceutical formulations also is
critically dependent on pH. And pH is also an important factor in
surface adsorption of many pharmaceutical peptides and
proteins.
[0007] Buffering agents that catalyze reactions that inactivate
and/or degrade one or more other ingredients, moreover, cannot be
used in pharmaceutical formulations. Buffers for pharmaceutical use
must have not only the buffer capacity required to maintain correct
pH, but also they must not buffer so strongly that their
administration deleteriously perturbs a subject's physiological pH.
Buffers for pharmaceutical formulations also must be compatible
with typically complex formulation processes. For instance, buffers
that sublime or evaporate, such as acetate and imidazole, generally
cannot be relied upon to maintain pH during lyophilization and in
the reconstituted lyophilization product. Other buffers that
crystallize out of the protein amorphous phase, such as sodium
phosphate, cannot be relied upon to maintain pH in processes that
require freezing.
[0008] Buffers used to maintain pH in pharmaceutical end-products
also must be not only effective at maintaining pH but also safe and
acceptable for administration to the subject. For instance, several
otherwise useful buffers, such as citrate at low or high
concentration and acetate at high concentration, are undesirably
painful when administered parenterally.
[0009] Some buffers have been found to be useful in the formulation
of pharmaceutical proteins, such as acetate, succinate, citrate,
histidine (imidazole), phosphate, and Tris. They all have
undesirable limitations and disadvantages. And they all have the
inherent disadvantage of being an additional ingredient in the
formulation, which complicates the formulation process, poses a
risk of deleteriously affecting other ingredients, stability,
shelf-life, and acceptability to the end user.
[0010] There is a need, therefore, for additional and improved
methods of maintaining pH in the production and formulation of
pharmaceuticals and in pharmaceutical compositions, particularly in
the production and formulation of biopharmaceutical proteins and in
biopharmaceutical protein compositions.
SUMMARY
[0011] Therefore, it is among the various objects and aspects of
the invention to provide, in certain of the preferred embodiments,
protein formulations comprising a protein, particularly
pharmaceutically acceptable formulations comprising a
pharmaceutical protein, that are buffered by the protein itself,
that do not require additional buffering agents to maintain a
desired pH, and in which the protein is substantially the only
buffering agent (i.e., other ingredients, if any, do not act
substantially as buffering agents in the formulation).
[0012] In this regard and others, it is among the various objects
and aspects of the invention to provide, in certain preferred
embodiments, self-buffering formulations of a protein, particularly
of a pharmaceutical protein, characterized in that the
concentration of the formulated protein provides a desired buffer
capacity.
[0013] It is further among the various objects and aspects of the
invention to provide, in certain of the particularly preferred
embodiments, self-buffering protein formulations, particularly
pharmaceutical protein formulations, in which the total salt
concentration is less than 150 mM.
[0014] It is further among the various objects and aspects of the
invention to provide, in certain of the particularly preferred
embodiments, self-buffering protein formulations, particularly
pharmaceutical protein formulations, that further comprise one or
more polyols and/or one or more surfactants.
[0015] It is also further among the various objects and aspects of
the invention to provide, in certain of the particularly preferred
embodiments, self-buffering formulations comprising a protein,
particularly a pharmaceutical protein, in which the total salt
concentration is less than 150 mM, that further comprise one or
more excipients, including but not limited to, pharmaceutically
acceptable salts; osmotic balancing agents (tonicity agents);
surfactants, polyols, anti-oxidants; antibiotics; antimycotics;
bulking agents; lyoprotectants; anti-foaming agents; chelating
agents; preservatives; colorants; and analgesics.
[0016] It is additionally among the various objects and aspects of
the invention to provide, in certain preferred embodiments,
self-buffering protein formulations, particularly pharmaceutical
protein formulations, that comprise, in addition to the protein,
one or more other pharmaceutically active agents.
[0017] Various additional aspects and embodiments of the invention
are illustratively described in the following numbered paragraphs.
The invention is described by way of reference to each of the items
set forth in the paragraphs, individually and/or taken together in
any combination. Applicant specifically reserves the right to
assert claims based on any such combination.
[0018] 1. A composition according to any of the following, wherein
the composition has been approved for pharmaceutical use by a
national or international authority empowered by law to grant such
approval preferably the European Agency for the Evaluation of
Medical Products, Japan's Ministry of Health, Labor and Welfare,
China's State Drug Administration, United States Food and Drug
Administration, or their successor(s) in this authority,
particularly preferably the United States Food and Drug
Administration or its successor(s) in this authority.
[0019] 2. A composition according to any of the foregoing or the
following, wherein the composition is produced in accordance with
good manufacturing practices applicable to the production of
pharmaceuticals for use in humans.
[0020] 3. A composition according to any of the foregoing or the
following, comprising a protein, the protein having a buffer
capacity per unit volume per pH unit of at least that of
approximately: 2.0 or 3.0 or 4.0 or 5.0 or 6.50 or 8.00 or 10.0 or
15.0 or 20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150
or 200 or 250 or 300 or 350 or 400 or 500 mM sodium acetate buffer
in pure water over the range of pH 5.0 to 4.0 or pH 5.0 to 5.5,
preferably as determined in accordance with the methods described
in Example 1 and 2, particularly preferably at least 2.0 mM,
especially particularly preferably at least 3.0 mM, very especially
particularly preferably at least 4.0 mM or at least 5.0 mM,
especially particularly preferably at least 7.5 mM, particularly
preferably at least 10 mM, preferably at least 20 mM.
[0021] 4. A composition according to any of the foregoing or the
following wherein, exclusive of the buffer capacity of the protein,
the buffer capacity per unit volume per pH unit of the composition
is equal to or less than that of 1.0 or 1.5 or 2.0 or 3.0 or 4.0 or
5.0 mM sodium acetate buffer in pure water over the range of pH 4.0
to 5.0 or pH 5.0 to 5.5, preferably as determined in accordance
with the methods described in Example 1 and 2, particularly
preferably less than that of 1.0 mM, very especially particularly
preferably less than that of 2.0 mM, especially particularly
preferably less than that of 2.5 mM, particularly preferably less
than that of 3.0 mM, preferably less than that of 5.0 mM.
[0022] 5. A composition according to any of the foregoing or the
following comprising a protein wherein over the range of plus or
minus 1 pH unit from the pH of the composition, the buffer capacity
of the protein is at least approximately: 1.00 or 1.50 or 1.63 or
2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 15.0 or
20.0 or 30.0 or 40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or
250 or 300 or 350 or 400 or 500 or 700 or 1,000 mEq per liter per
pH unit, preferably at least approximately 1.00, particularly
preferably 1.50, especially particularly preferably 1.63, very
especially particularly preferably 2.00, very highly especially
particularly preferably 3.00, very especially particularly
preferably 5.0, especially particularly preferably 10.0,
particularly preferably 20.0.
[0023] 6. A composition according to any of the foregoing or the
following comprising a protein wherein over the range of plus or
minus 1 pH unit from the pH of the composition, exclusive of the
protein, the buffer capacity per unit volume per pH unit of the
composition is equal to or less than that of 0.50 or 1.00 or 1.50
or 2.00 or 3.00 or 4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 20.0 or
25.0 mM sodium acetate buffer in pure water over the range pH 5.0
to 4.0 or pH 5.0 to 5.5, particularly preferably determined in
accordance with Example 1 and/or Example 2.
[0024] 7. A composition according to any of the foregoing or the
following, wherein over a range of plus or minus 1 pH unit from a
desired pH, the protein provides at least approximately 55%, 60%,
65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of the
buffer capacity of the composition, preferably at least
approximately 75%, particularly preferably at least approximately
85%, especially particularly preferably at least approximately 90%,
very especially particularly preferably at least approximately 95%,
very highly especially particularly preferably at least
approximately 99% of the buffer capacity of the composition.
[0025] 8. A composition according to any of the foregoing or the
following, wherein the concentration of the protein is between
approximately: 20 and 400, or 20 and 300, or 20 and 250, or 20 and
200, or 20 and 150 mg/ml, preferably between approximately 20 and
400 mg/ml, particularly preferably between approximately 20 and
250, especially particularly between approximately 20 and 150
mg/ml.
[0026] 9. A composition according to any of the foregoing or the
following, wherein the pH maintained by the buffering action of the
protein is between approximately: 3.5 and 8.0, or 4.0 and 6.0, or
4.0 and 5.5, or 4.0 and 5.0, preferably between approximately 3.5
and 8.0, especially particularly preferably approximately 4.0 and
5.5.
[0027] 10. A composition according to any of the foregoing or the
following, wherein the salt concentration is less than: 150 mM or
125 mM or 100 mM or 75 mM or 50 mM or 25 mM, preferably 150 mM,
particularly preferably 125 mM, especially preferably 100 mM, very
particularly preferably 75 mM, particularly preferably 50 mM,
preferably 25 mM.
[0028] 11. A composition according to any of the foregoing or the
following, further comprising one or more pharmaceutically
acceptable salts; polyols; surfactants; osmotic balancing agents;
tonicity agents; anti-oxidants; antibiotics; antimycotics; bulking
agents; lyoprotectants; anti-foaming agents; chelating agents;
preservatives; colorants; analgesics; or additional pharmaceutical
agents.
[0029] 12. A composition according to any of the foregoing or the
following, comprising one or more pharmaceutically acceptable
polyols in an amount that is hypotonic, isotonic, or hypertonic,
preferably approximately isotonic, particularly preferably
isotonic, especially preferably any one or more of sorbitol,
mannitol, sucrose, trehalose, or glycerol, particularly especially
preferably approximately 5% sorbitol, 5% mannitol, 9% sucrose, 9%
trehalose, or 2.5% glycerol, very especially in this regard 5%
sorbitol, 5% mannitol, 9% sucrose, 9% trehalose, or 2.5%
glycerol.
[0030] 13. A composition according to any of the foregoing or the
following, further comprising a surfactant, preferably one or more
of polysorbate 20, polysorbate 80, other fatty acid esters of
sorbitan, polyethoxylates, and poloxamer 188, particularly
preferably polysorbate 20 or polysorbate 80, preferably
approximately 0.001 to 0.1% polysorbate 20 or polysorbate 80, very
preferably approximately 0.002 to 0.02% polysorbate 20 or
polysorbate 80, especially 0.002 to 0.02% polysorbate 20 or
polysorbate 80.
[0031] 14. A composition according to any of the foregoing or the
following, wherein the protein is a pharmaceutical agent and the
composition is a sterile formulation thereof suitable for treatment
of a non-human or a human subject.
[0032] 15. A composition according to any of the foregoing or the
following, wherein the protein is a pharmaceutical agent effective
to treat a disease and the composition is a sterile formulation
thereof suitable for administration to a subject for treatment
thereof.
[0033] 16. A composition according to any of the foregoing or the
following, wherein the protein does not induce a significantly
deleterious antigenic response following administration to a
subject.
[0034] 17. A composition according to any of the foregoing or the
following, wherein the protein does not induce a significantly
deleterious immune response following administration to a
subject.
[0035] 18. A composition according to any of the foregoing or the
following, wherein the protein is a human protein.
[0036] 19. A composition according to any of the foregoing or the
following, wherein the protein is a humanized protein.
[0037] 20. A method according to any of the foregoing or the
following, wherein the protein is an antibody, preferably an IgA,
IgD, IgE, IgG, or IgM antibody, particularly preferably an IgG
antibody, very particularly preferably an IgG1, IgG2, IgG3, or IgG4
antibody, especially an IgG2 antibody.
[0038] 21. A composition according to any of the foregoing or the
following, wherein the protein comprises a: Fab fragment, Fab.sub.2
fragment, Fab.sub.3 fragment, Fc fragment, scFv fragment,
bis-scFv(s) fragment, minibody, diabody, triabody, tetrabody, VhH
domain, V-NAR domain, V.sub.H domain, V.sub.L domain, camel Ig, Ig
NAR, or peptibody, or a variant, derivative, or modification of any
of the foregoing.
[0039] 22. A composition according to any of the foregoing or the
following, wherein the protein comprises an Fc fragment or a part
thereof or a derivative or variant of an Fc fragment or part
thereof.
[0040] 23. A composition according to any of the foregoing or the
following, wherein the protein comprises a first binding moiety of
a pair of cognate binding moieties, wherein the first moiety binds
the second moiety specifically.
[0041] 24. A composition according to any of the foregoing or the
following, wherein the protein comprises (a) an Fc fragment or a
part thereof or a derivative or variant of an Fc fragment or part
thereof, and (b) a first binding moiety of a pair of cognate
binding moieties.
[0042] 25. A composition according to any of claims 1, 5, 7, 9, 11,
13, or 14, wherein the protein is selected from the group
consisting of proteins that bind specifically to one or more CD
proteins, HER receptor family proteins, cell adhesion molecules,
growth factors, nerve growth factors, fibroblast growth factors,
transforming growth factors (TGF), insulin-like growth factors,
osteoinductive factors, insulins and insulin-related proteins,
coagulation and coagulation-related proteins, colony stimulating
factors (CSFs), other blood and serum proteins blood group
antigens; receptors, receptor-associated proteins, growth hormone
receptors, T-cell receptors; neurotrophic factors. neurotrophins,
relaxins, interferons, interleukins, viral antigens, lipoproteins,
integrins, rheumatoid factors, immunotoxins, surface membrane
proteins, transport proteins, homing receptors, addressing,
regulatory proteins, and immunoadhesins,
[0043] 26. A composition according to any of the foregoing or the
following, wherein the protein is selected from the group
consisting of: OPGL specific binding proteins, myostatin specific
binding proteins, IL-4 receptor specific binding proteins, IL1-R1
specific binding proteins, Ang2 specific binding proteins,
NGF-specific binding proteins, CD22 specific binding proteins,
IGF-1 receptor specific binding proteins, B7RP-1 specific binding
proteins, IFN gamma specific binding proteins, TALL-1 specific
binding proteins, stem cell factors, Flt-3 ligands, and IL-17
receptors.
[0044] 27. A composition according to any of the foregoing or the
following, wherein the protein is selected from the group
consisting of proteins that bind specifically to one or more of:
CD3, CD4, CD8, CD19, CD20, CD34; HER2, HER3, HER4, the EGF
receptor; LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, alpha v/beta 3
integrin; vascular endothelial growth factor ("VEGF"); growth
hormone, thyroid stimulating hormone, follicle stimulating hormone,
luteinizing hormone, growth hormone releasing factor, parathyroid
hormone, mullerian-inhibiting substance, human macrophage
inflammatory protein (MIP-1-alpha), erythropoietin (EPO), NGF-beta,
platelet-derived growth factor (PDGF), aFGF, bFGF, epidermal growth
factor (EGF), TGF-alpha, TGF-beta1, TGF-beta2, TGF-beta3,
TGF-beta4, TGF-beta5, IGF-I, IGF-II, des(1-3)-IGF-I (brain IGF-I),
insulin, insulin A-chain, insulin B-chain, proinsulin, insulin-like
growth factor binding proteins;, such as, among others, factor
VIII, tissue factor, von Willebrands factor, protein C,
alpha-1-antitrypsin, plasminogen activators, such as urokinase and
tissue plasminogen activator ("t-PA"), bombazine, thrombin, and
thrombopoietin; M-CSF, GM-CSF, G-CSF, albumin, IgE, flk2/flt3
receptor, obesity (OB) receptor, bone-derived neurotrophic factor
(BDNF), NT-3, NT-4, NT-5, NT-6); relaxin A-chain, relaxin B-chain,
prorelaxin; interferon-alpha, -beta, and -gamma; IL-1 to IL-10;
AIDS envelope viral antigen; calcitonin, glucagon, atrial
natriuretic factor, lung surfactant, tumor necrosis factor-alpha
and -beta, enkephalinase, RANTES, mouse gonadotropin-associated
peptide, Dnase, inhibin, and activin; protein A or D, bone
morphogenetic protein (BMP), superoxide dismutase, decay
accelerating factor (DAF).
[0045] 28. A composition according to any of the foregoing or the
following, wherein the protein is selected from the group
consisting of: Actimmune (Interferon-gamma-1b), Activase
(Alteplase), Aldurazme (Laronidase), Amevive (Alefacept), Avonex
(Interferon beta-1a), BeneFIX (Nonacog alfa), Beromun (Tasonermin),
Beatseron (Interferon-beta-1b), BEXXAR (Tositumomab), Tev-Tropin
(Somatropin), Bioclate or RECOMBINATE (Recombinant), CEREZME
(Imiglucerase), ENBREL (Etanercept), Eprex (epoetin alpha),
EPOGEN/Procit (Epoetin alfa), FABRAZYME (Agalsidase beta),
Fasturtec/Elitek ELITEK ((Rasburicase), FORTEO (Teriparatide),
GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon,
rDNA origin), GONAL-F (follitropin alfa), KOGENATE FS (Octocog
alfa), HERCEPTIN (Trastuzumab), HUMATROPE (SOMATROPIN), HUMIRA
(Adalimumab), Insulin in Solution, INFERGEN.RTM. (Interferon
alfacon-1), KINERET.RTM. (anakinra), Kogenate FS (Antihemophilic
Factor), LEUKIN (SARGRAMOSTIM Recombinant human
granulocyte-macrophage colony stimulating factor (rhuGM-CSF)),
CAMPATH (Alemtuzumab), RITUXAN.RTM. (Rituximab), TNKase
(Tenecteplase), MYLOTARG (gemtuzumab ozogamicin), NATRECOR
(nesiritide), ARANESP (darbepoetin alfa), NEULASTA (pegfilgrastim),
NEUMEGA (oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES
(Somatropin), NOVOSEVEN (Eptacog alfa), NUTROPIN AQ (somatropin),
Oncaspar (pegaspargase), ONTAK (denileukin diftitox), ORTHOCLONE
OKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS
(peginterferon alfa-2a), PROLEUKIN (Aldesleukin), PULMOZYME
(dornase alfa), Retavase (Reteplase), REBETRON Combination Therapy
containing REBETOL.RTM. (Ribavirin) and INTRON.RTM. A (Interferon
alfa-2b), REBIF (interferon beta-1a), REFACTO (Antihemophilic
Factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO
(abciximab)ROFERON.RTM.-A (Interferon alfa-2a), SIMULECT
(baasiliximab), SOMAVERT (Pegivisomant), SYNAGIS.RTM.
(palivizumab), Stemben (Ancestim, Stem cell factor), THYROGEN,
INTRON.RTM. A (Interferon alfa-2b), PEG-INTRON.RTM. (Peginterferon
alfa-2b), XIGRIS.RTM. (Drotrecogin alfa activated), XOLAIR.RTM.
(Omalizumab), ZENAPAX.RTM. (daclizumab), and ZEVALIN.RTM.
(Ibritumomab Tiuxetan).
[0046] 29. A composition according to any of the foregoing or the
following, wherein the protein is Ab-hCD22 or a fragment thereof,
or a variant, derivative, or modification of Ab-hCD22 or of a
fragment thereof; Ab-hIL4R or a fragment thereof, or a variant,
derivative, or modification of Ab-hIL4R or of a fragment thereof;
Ab-hOPGL or a fragment thereof, or a variant, derivative, or
modification of Ab-hOPGL or of a fragment thereof, or Ab-hB7RP1 or
a fragment thereof, or a variant, derivative, or modification of
Ab-hB7RP1 or of a fragment thereof.
[0047] 30. A composition according to any of the foregoing or the
following, wherein the protein is: Ab-hCD22 or Ab-hIL4R or Ab-hOPGL
or Ab-hB7RP1.
[0048] 31. A composition according to any of the foregoing or the
following comprising a protein and a solvent, the protein having a
buffer capacity per unit volume per pH unit of at least that of 4.0
mM sodium acetate in water over the range of pH 4.0 to 5.0 or pH
5.0 to 5.5, particularly as determined by the methods described in
Examples 1 and 2, wherein the buffer capacity per unit volume of
the composition exclusive of the protein is equal to or less than
that of 2.0 mM sodium acetate in water over the same ranges
preferably determined in the same way.
[0049] 32. A composition according to any of the foregoing or the
following comprising a protein and a solvent, wherein at the pH of
the composition the buffer capacity of the protein is at least 1.63
mEq per liter for a pH change of the composition of plus or minus 1
pH unit wherein the buffer capacity of the composition exclusive of
the protein is equal to or less than 0.81 mEq per liter at the pH
of the composition for a pH change of plus or minus 1 pH unit.
[0050] 33. A lyophilate which upon reconstitution provides a
composition in accordance with any of the foregoing or the
following.
[0051] 34. A kit comprising in one or more containers a composition
or a lyophilate in accordance with any of the foregoing or the
following, and instructions regarding use thereof.
[0052] 35. A process for preparing a composition or a lyophilate
according to any of the foregoing or the following, comprising
removing residual buffer using a counter ion.
[0053] 36. A process for preparing a composition or a lyophilate
according to any of the foregoing or the following, comprising
removing residual buffer using any one or more of the following in
the presence of a counter ion: chromatography, dialysis, and/or
tangential flow filtration.
[0054] 37. A process for preparing a composition or a lyophilate
according to any of the foregoing or the following, comprising
removing residual buffer using tangential flow filtration.
[0055] 38. A process for preparing a composition or a lyophilate
according to any of the foregoing or the following comprising a
step of dialysis against a solution at a pH below that of the
preparation, and, if necessary, adjusting the pH thereafter by
addition of dilute acid or dilute base.
[0056] 39. A method for treating a subject comprising administering
to a subject in an amount and by a route effective for treatment a
composition according to any of the foregoing or the following,
including a reconstituted lyophilate.
BRIEF DESCRIPTION OF THE FIGURES
[0057] FIG. 1 depicts titration data and buffer capacity as a
function of concentration for sodium acetate standard buffers over
the range from pH 5.0 to 4.0. Panel A is a graph that depicts the
pH change upon acid titration of several different concentrations
of a standard sodium acetate buffer, as described in Example 1. pH
is indicated on the vertical axis. The amount of acid added to each
solution is indicated on the horizontal axis in microequivalents of
HCl added per ml of solution (.mu.Eq/ml). The linear least squares
trend lines are depicted for each dataset. Acetate concentrations
are indicated in the inset. Panel B is a graph that depicts the
buffer capacity of the acetate buffers over the acidic pH range as
determined from the titration data depicted in Panel A, as
described in Example 1. Buffer capacity is indicated on the
vertical axis as microequivlents of acid per ml of buffer solution
per unit change in pH (.mu.Eq/ml-pH). Acetate concentration is
indicated on the horizontal axis in mM.
[0058] FIG. 2 depicts titration data and buffer capacity as a
function of concentrations for sodium acetate standard buffers over
the range from pH 5.0 to 5.5. Panel A is a graph that depicts the
pH change upon base titration of several different concentration of
a standard sodium acetate buffer, as described in Example 2. pH is
indicated on the vertical axis. The amount of base added to each
solution is indicated on the horizontal axis in microequivalents of
NaOH added per ml of solution (.mu.Eq/ml). The linear least squares
trend lines are depicted for each dataset. Acetate concentrations
are indicated in the inset. Panel B is a graph that depicts the
buffer capacity of the acetate buffers over the basic pH range as
determined from the titration data depicted in Panel A and
described in Example 2 Buffer capacity is indicated on the vertical
axis as microequivlents of base per ml of buffer solution per unit
change in pH (.mu.Eq/ml-pH). Acetate concentration is indicated on
the horizontal axis in mM.
[0059] FIG. 3 depicts the determination of acetate concentration in
acetate buffer standards, as described in Example 3. The graph
shows a standard curve for the determinations, with peak area
indicated on the vertical axis and the acetate concentration
indicated on the horizontal axis. The nominal and the measured
amounts of acetate in the solutions used for the empirical
determination of buffer capacity are tabulated below the graph.
[0060] FIG. 4 is a graph that depicts the pH change upon acid
titration of several different concentrations of Ab-hOPGL over the
range of pH 5.0 to 4.0, as described in Example 4. pH is indicated
on the vertical axis. The amount of acid added to the solutions is
indicated on the horizontal axis in microequivalents of HCl added
per ml of buffer solution (.mu.Eq/ml). The linear least squares
trend lines are depicted for each dataset. Ab-hOPGL concentrations
are indicated in the inset.
[0061] FIG. 5 is a graph that depicts the pH change upon base
titration of several different concentrations of Ab-hOPGL over the
range 5.0 to 6.0, as described in Example 5. pH is indicated on the
vertical axis. The amount of base added to the solutions is
indicated on the horizontal axis in microequivalents of NaOH added
per ml of buffer solution (.mu.Eq/ml). The linear least squares
trend lines are depicted for each dataset. Ab-hOPGL concentrations
are indicated in the inset.
[0062] FIG. 6 shows the residual acetate levels in Ab-hOPGL
solutions used for determining buffer capacity. The graph shows the
standard curve used for the acetate determinations as described in
Example 6. The nominal and the experimentally measured acetate
concentrations in the solutions are tabulated below the graph.
[0063] FIG. 7 is a graph depicting the buffer capacity of Ab-hOPGL
plus or minus residual acetate in the pH range 5.0 to 4.0. The data
were obtained as described in Example 7. The upper line shows
Ab-hOPGL buffer capacity with residual acetate. The lower line
shows Ab-hOPGL buffer capacity adjusted for residual acetate. The
vertical axis indicates buffer capacity in microequivalents of acid
per ml of Ab-hOPGL solution per unit of pH (.mu.Eq/ml-pH). The
horizontal axis indicates the concentration of Ab-hOPGL in mg/ml.
The buffer capacities of different concentrations of standard
acetate buffers as described in Example 1 are shown as horizontal
lines. The concentrations of the buffers are indicated above the
lines.
[0064] FIG. 8 is a graph depicting the buffer capacity of Ab-hOPGL
plus or minus residual acetate in the basic pH range pH 5.0 to 6.0.
The data were obtained as described in Example 8. The upper line
depicts Ab-hOPGL buffer capacity with residual acetate. The lower
line depicts Ab-hOPGL buffer capacity adjusted for residual
acetate. The vertical axis indicates buffer capacity in
microequivalents of base added per ml of buffer solution per unit
of pH (.mu.Eq/ml-pH). The horizontal axis indicates the
concentration of Ab-hOPGL in mg/ml. The buffer capacities of
several concentrations of standard sodium acetate buffers as
described in Example 2 are indicated by horizontal lines. The
acetate concentrations are indicated above each line.
[0065] FIG. 9 depicts, in a pair of charts, pH and Ab-hOPGL
stability in self-buffering and conventionally buffered
formulations. Panel A depicts the stability of self-buffered
Ab-hOPGL, Ab-hOPGL formulated in acetate buffer, and Ab-hOPGL
formulated in glutamate as a function of storage time at 4.degree.
C. over a period of six months. The vertical axis indicates
Ab-hOPGL stability in percent Ab-hOPGL monomer determined by
SE-HPLC. Storage time is indicated on the horizontal axis. Panel B
depicts the pH of the same three formulations measured over the
same period of time. The determinations of protein stability and
the measurements of pH are described in Example 9.
[0066] FIG. 10 depicts titration curves and buffer capacities for
several concentrations of self-buffering Ab-hB7RP1 formulations
over the range of pH 5.0 to 4.0. Panel A shows the titration data.
pH is indicated on the vertical axis. The amount of acid added to
the solutions is indicated on the horizontal axis in
microequivalents of HCl added per ml of buffer solution
(.mu.Eq/ml). The linear least squares trend lines are depicted for
each dataset. The Ab-hB7RP1 concentrations are indicated in the
inset. Panel B depicts the buffer capacities of Ab-hB7RP1
formulations. The upper line shows the buffer capacities for the
formulations including the contribution of residual acetate. The
lower line shows the buffer capacities for formulations after
subtracting the contribution of residual acetate based on SE-HPLC
determinations as described in Example 3. Linear least squares
trend lines are shown for the two data sets. The vertical axis
indicates buffer capacity in microequivalents of acid per ml of
buffer solution per unit of pH (.mu.Eq/ml-pH). The concentration of
Ab-hB7RP1 is indicated on the horizontal axis in mg/ml. The buffer
capacities of several concentrations of standard sodium acetate
buffers as described in Example 1 are shown by dashed horizontal
lines. The acetate buffer concentration are shown below each line.
The results were obtained as described in Example 10.
[0067] FIG. 11 depicts titration curves and buffer capacities for
several concentrations of self-buffering Ab-hB7RP1 formulations
over the range of pH 5.0 to 6.0. Panel A shows the titration data.
pH is indicated on the vertical axis. The amount of base added to
the solutions is indicated on the horizontal axis in
microequivalents of NaOH added per ml of buffer solution
(.mu.Eq/ml). The linear least squares trend lines are depicted for
each dataset. The Ab-hB7RP1 concentrations are indicated in the
inset. Panel B depicts the buffer capacities of Ab-hB7RP1
formulations. The upper line shows the buffer capacities for the
formulations containing residual acetate. The lower line shows the
buffer capacities for formulations adjusted to remove the
contribution of residual acetate. Linear least squares trend lines
are shown for the two data sets. The vertical axis indicates buffer
capacity in microequivalents of base per ml of buffer solution per
unit of pH (.mu.Eq/ml-pH). The concentration of Ab-hB7RP1 is
indicated on the horizontal axis in mg/ml. The buffer capacities of
several concentrations of standard sodium acetate buffers as
described in Example 2 are shown by dashed horizontal lines. The
acetate buffer concentrations are shown above each line. The
results were obtained as described in Example 11.
[0068] FIG. 12 depicts Ab-hB7RP1 stability in self-buffering and
conventionally buffered formulations at 4.degree. C. and 29.degree.
C. Panel A depicts the stability of self-buffered Ab-hB7RP1,
Ab-hB7RP1 formulated in acetate buffer, and Ab-hB7RP1 formulated in
glutamate as a function of storage at 4.degree. C. over a period of
six months. The vertical axis depicts Ab-hB7RP1 monomer in the
samples determined by SE-HPLC. Time is indicated on the horizontal
axis. Panel B depicts the stability of the same three formulations
as a function of storage at 29.degree. C. over the same period of
time. Axes in Panel B are the same as in Panel A. The
determinations of protein stability by HPLC-SE are described in
Example 12.
[0069] FIG. 13 depicts pH stability in self buffer formulations of
Ab-hB7RP1 at 4.degree. C. and 29.degree. C. The vertical axis
indicates pH. Time, in weeks, is indicated on the horizontal axis.
Temperatures of the datasets are indicated in the inset. The data
were obtained as described in Example 13.
[0070] FIG. 14 depicts the buffer capacity of self-buffering
formulations of Ab-hCD22 as a function of Ab-hCD22 concentration
over the range of pH 4.0 to 6.0. Panel A depicts the buffer
capacities of self-buffering Ab-hCD22 formulations as a function of
Ab-hCD22 concentration over the range of pH 4.0 to 5.0. Panel B
depicts the buffer capacities of self-buffering Ab-hCD22
formulations as a function of concentration over the range of pH
5.0 to 6.0. In both panels the vertical axis indicates buffer
capacity in microequivalents of base per ml of buffer solution per
unit of pH (.mu.Eq/ml-pH), and the horizontal axis indicates
Ab-hCD22 concentrations in mg/ml. For reference, the buffer
capacity of 10 mM sodium acetate as described in Example 1 is shown
in both panels by a dashed horizontal line. The results shown in
the Figure were obtained as described in Example 14.
[0071] FIG. 15 depicts titration curves and buffer capacities for
several concentrations of self-buffering Ab-hIL4R formulations over
the range of pH 5.0 to 4.0. Panel A shows the titration data. pH is
indicated on the vertical axis. The amount of acid added to the
solutions is indicated on the horizontal axis in microequivalents
of HCl added per ml of buffer solution (.mu.Eq/ml). The linear
least squares trend lines are depicted for each dataset. The
Ab-hIL4R concentrations are indicated in the inset. Panel B depicts
the buffer capacities of Ab-hIL4R as a function of concentration.
The linear least squares trend line is shown for the dataset. The
vertical axis indicates buffer capacity in microequivalents of base
per ml of buffer solution per unit of pH (.mu.Eq/ml-pH). The
concentration of Ab-hIL4R is indicated on the horizontal axis in
mg/ml. The buffer capacities of several concentrations of standard
sodium acetate buffers as described in Example 1 are shown by
dashed horizontal lines. The acetate buffer concentrations are
shown above each line. The results were obtained as described in
Example 15.
[0072] FIG. 16 depicts titration curves and buffer capacities for
several concentrations of self-buffering Ab-hIL4R formulations over
the range of pH 5.0 to 6.0. Panel A shows the titration data. pH is
indicated on the vertical axis. The amount of base added to the
solutions is indicated on the horizontal axis in microequivalents
of NaOH added per ml of buffer solution (.mu.Eq/ml). The linear
least squares trend lines are depicted for each dataset. The
Ab-hIL4R concentrations are indicated in the inset. Panel B depicts
the buffer capacities of Ab-hIL4R as a function of concentration.
The linear least squares trend line is shown for the dataset. The
vertical axis indicates buffer capacity in microequivalents of base
per ml of buffer solution per unit of pH (.mu.Eq/ml-pH). The
concentration of Ab-hIL4R is indicated on the horizontal axis in
mg/ml. The buffer capacities of several concentrations of standard
sodium acetate buffers as described in Example 2 are shown by
dashed horizontal lines. The acetate buffer concentrations are
shown above each line. The results were obtained as described in
Example 16.
[0073] FIG. 17 depicts Ab-hIL4R and pH stability in acetate
buffered and self-buffered formulations of Ab-hIL4R at 37.degree.
C. as a function of time. Panel A is a bar graph showing Ab-hIL4R
stability over four weeks at 37.degree. C. The vertical axis
indicates stability in percent monomeric Ab-hIL4R as determined by
SE-HPLC. The horizontal axis indicates storage time in weeks. The
insert identifies the data for the acetate and for the
self-buffered formulations. Panel B shows the pH stability of the
same formulations for the same conditions and time periods. The pH
is indicated on the vertical axis. Storage time in weeks is
indicated on the horizontal axis. Data for the acetate and
self-buffered formulations are indicated in the inset. The data
were obtained as described in Example 17.
GLOSSARY
[0074] The meanings ascribed to various terms and phrases as used
herein are illustratively explained below.
[0075] "A" or "an" herein means "at least one;" "one or more than
one."
[0076] "About," unless otherwise stated explicitly herein, means V
20%. For instance about 100 herein means 80 to 120, about 5 means 4
to 6, about 0.3 means 0.24 to 0.36, and about 60% means 48% to 72%
(not 40% to 80%).
[0077] "Agonist(s)" means herein a molecular entity that is
different from a corresponding stimulatory ligand but has the same
stimulatory effect. For instance (although agonists work through
other mechanisms), for a hormone that stimulates an activity by
binding to a corresponding hormone receptor, an agonist is a
chemically different entity that binds the hormone receptor and
stimulates its activity.
[0078] "Antagonist(s)" means herein a molecular entity that is
different from a corresponding ligand and has an opposite effect.
For instance (although antagonists work through other mechanisms),
one type of antagonist of a hormone that stimulates an activity by
binding to a corresponding hormone receptor is a chemical entity
that is different from the hormone and binds the hormone receptor
but does not stimulate the activity engendered by hormone binding,
and by this action inhibits the effector activity of the
hormone.
[0079] "Antibody(s)" is used herein in accordance with its ordinary
meaning in the biochemical and biotechnological arts.
[0080] Among antibodies within the meaning of the term as it is
used herein, are those isolated from biological sources, including
monoclonal and polyclonal antibodies, antibodies made by
recombinant DNA techniques (also referred to at times herein as
recombinant antibodies), including those made by processes that
involve activating an endogenous gene and those that involve
expression of an exogenous expression construct, including
antibodies made in cell culture and those made in transgenic plants
and animals, and antibodies made by methods involving chemical
synthesis, including peptide synthesis and semi-synthesis. Also
within the scope of the term as it is used herein, except as
otherwise explicitly set forth, are chimeric antibodies and hybrid
antibodies, among others.
[0081] The prototypical antibody is a tetrameric glycoprotein
comprised of two identical light chain-heavy chain dimers joined
together by disulfide bonds. There are two types of vertebrate
light chains, kappa and lambda. Each light chain is comprised of a
constant region and a variable region. The two light chains are
distinguished by constant region sequences. There are five types of
vertebrate heavy chains: alpha, delta, epsilon, gamma, and mu. Each
heavy chain is comprised of a variable region and three constant
regions. The five heavy chain types define five classes of
vertebrate antibodies (isotypes): IgA, IgD, IgE, IgG, and IgM. Each
isotype is made up of, respectively, (a) two alpha, delta, epsilon,
gamma, or mu heavy chains, and (b) two kappa or two lambda light
chains. The heavy chains in each class associate with both types of
light chains; but, the two light chains in a given molecule are
both kappa or both lambda. IgD, IgE, and IgG generally occur as
"free" heterotetrameric glycoproteins. IgA and IgM generally occur
in complexes comprising several IgA or several IgM heterotetramers
associated with a "J" chain polypeptide. Some vertebrate isotypes
are classified into subclasses, distinguished from one another by
differences in constant region sequences. There are four human IgG
subclasses, IgG1, IgG2, IgG3, and IgG4, and two IgA subclasses,
IgA1 and IgA2, for example. All of these and others not
specifically described above are included in the meaning of the
term "antibody(s)" as used herein.
[0082] The term "antibody(s)" further includes amino acid sequence
variants of any of the foregoing as described further elsewhere
herein.
[0083] "Antibody-derived" as used herein means any protein produced
from an antibody, and any protein of a design based on an antibody.
The term includes in its meaning proteins produced using all or
part of an antibody, those comprising all or part of an antibody,
and those designed in whole or in part on the basis of all or part
of an antibody. "Antibody-derived" proteins include, but are not
limited to, Fc, Fab, and Fab.sub.2 fragments and proteins
comprising the same, V.sub.H domain and V.sub.L domain fragments
and proteins comprising the same, other proteins that comprise a
variable and/or a constant region of an antibody, in whole or in
part, scFv(s) intrabodies, maxibodies, minibodies, diabodies, amino
acid sequence variants of the foregoing, and a variety of other
such molecules, including but not limited to others described
elsewhere herein.
[0084] "Antibody-related" as used herein means any protein or
mimetic resembling in its structure, function, or design an
antibody or any part of an antibody. Among "antibody-related"
proteins as the term is used herein are "antibody-derived" proteins
as described above. It is to be noted that the terms
"antibody-derived" and "antibody-related" substantially overlap;
both terms apply to many such proteins. Examples of
"antibody-related" proteins, without implying limitation in this
respect, are peptibodies and receptibodies. Other examples of
"antibody-related" proteins are described elsewhere herein.
[0085] "Antibody polypeptide(s)" as used herein, except as
otherwise noted, means a polypeptide that is part of an antibody,
such as a light chain polypeptide, a heavy chain polypeptide and a
J chain polypeptide, to mention a few examples, including among
others fragments, derivatives, and variants thereof, and related
polypeptides.
[0086] "Approximately" unless otherwise noted means the same as
about.
[0087] "Binding moiety(s)" means a part of a molecule or a complex
of molecules that binds specifically to part of another molecule or
complex of molecules. The binding moiety may be the same or
different from the part of the molecule or complex of molecules to
which it binds. The binding moiety may be all of a molecule or
complex of molecules as well.
[0088] "Binds specifically" is used herein in accordance with its
ordinary meaning in the art and means, except as otherwise noted,
that binding is stronger with certain specific moieties than it is
to other moieties in general, that it is stronger than non-specific
binding that may occur with a wide variety of moieties, and that
binding is selective for certain moieties and does not occur to as
strong an extent with others. In the extreme case of specific
binding, very strong binding occurs with a single type of moiety,
and there is no non-specific binding with any other moiety.
[0089] "Co-administer" means an administration of two or more
agents in conjunction with one another, including simultaneous
and/or sequential administration.
[0090] "Cognate(s)" herein means complementary, fitting together,
matching, such as, for instance, two jigsaw puzzles that fit one
another, the cylinder mechanism of a lock and the key that opens
it, the substrate binding site of an enzyme and the substrate of
the enzyme, and a target and target binding protein that binds
specifically thereto.
[0091] "Cognate binding moieties" herein means binding moieties
that bind specifically to one another. Typically, but not always,
it means a pair of binding moieties that bind specifically to one
another. The moieties responsible for highly selective binding of a
specific ligand and ligand receptor provide an illustrative example
of cognate binding moieties. Another example is provided by the
moieties that binds an antigen and an antibody.
[0092] "Composition" means any composition of matter comprising one
or more constituents, such as a formulation.
[0093] "Comprised of" is a synonym of "comprising" (see below).
[0094] "Comprising" means including, without further qualification,
limitation, or exclusion as to what else may or may not be
included. For example, "a composition comprising x and y" means any
composition that contains x and y, no matter what else it may
contain. Likewise, "a method comprising x" is any method in which x
is carried out, no matter what else may occur.
[0095] "Concentration" is used herein in accordance with its
well-known meaning in the art to mean the amount of an item in a
given amount of a mixture containing the item, typically expressed
as a ratio. For example, concentration of a solute, such as a
protein in a solution, can be expressed in many ways, such as (but
not limited to): (A) Weight Percent (i)=weight of solute per 100
units of solvent volume; (B) Weight Percent (ii)=weight of solute
per 100 units of total weight; (C) Weight Percent (iii) weight of
solute per 100 units of solvent by weight; (D) Mass Percent=mass of
solute per 100 mass units of solution; (E) Mole Fraction=moles of
solute per total moles of all components; (F) Molarity=moles of
solute per liter of solution (i.e., solute plus solvent); (G)
Molality=moles of solute per Kg of solvent; and (H) Volume
Molality=moles of solute per liter of solvent.
[0096] "Control region(s)" is used herein in accordance with its
well-known meaning in the art, and except as noted otherwise,
refers to regions in DNA or proteins that are responsible for
controlling one or more functions or activities thereof. For
instance, "expression control region" with reference to the control
of gene expression, means the regions in DNA that are required for
transcription to occur properly and that are involved in regulating
when transcription occurs, how efficiently it occurs, when it is
stopped, and the like.
[0097] "De novo" is used herein in accordance with its well-known
meaning in the art, to denote something made from new. For
instance, a de novo amino acid sequence is one not derived from a
naturally occurring amino acid sequence, although, such a de novo
sequence may have similarities with a naturally occurring sequence.
De novo amino acid sequences can be generated, for instance, by a
priori design, by combinatorial methods, by selection methods. They
can be made, for example, by chemical synthesis, by semi-synthesis,
and by a variety of recombinant DNA techniques, all of which are
well know to those skilled in the art.
[0098] "Deleterious" means, as used herein, harmful. By way of
illustration, "deleterious" processes include, for example, harmful
effects of disease processes and harmful side effects of
treatments.
[0099] "Derivative(s)" is used herein to mean derived from, in
substance, form, or design, such as, for instance, a polypeptide
that is based on but differs from a reference polypeptide, for
instance, by alterations to its amino acid sequence, by fusion to
another polypeptide, or by covalent modification.
[0100] "Disease(s)" a pathology, a condition that deleteriously
affects health of a subject.
[0101] "Disorder(s)" a malediction, a condition that deleteriously
alters health.
[0102] "Dysfunction" means, as used herein, a disorder, disease, or
deleterious effect of an otherwise normal process.
[0103] "Effective amount" generally means an amount which provides
the desired local or systemic effect. For example, an effective
amount is an amount sufficient to effectuate a beneficial or
desired clinical result. The effective amount can be provided all
at once in a single administration or in fractional amounts that
provide the effective amount in several administrations. The
precise determination of what would be considered an effective
amount may be based on factors individual to each subject,
including their size, age, injury, and/or disease or injury being
treated, and amount of time since the injury occurred or the
disease began. One skilled in the art will be able to determine the
effective amount for a given subject based on these considerations
which are routine in the art. As used herein, "effective dose"
means the same as "effective amount."
[0104] "Effective route" generally means a route which provides for
delivery of an agent to a desired compartment, system, or location.
For example, an effective route is one through which an agent can
be administered to provide at the desired site of action an amount
of the agent sufficient to effectuate a beneficial or desired
clinical result.
[0105] "Endogenous" (such as endogenous gene) is used herein to
refer to, for instance, genes and other aspects of DNA, such as
control regions, that naturally occur in a genome and organism,
unless otherwise indicated.
[0106] "Exogenous" (such as exogenous gene), unless otherwise
indicated, is used herein generally to mean, for instance, DNA from
an outside source, such as DNA introduced to a cell and
incorporated into its genome.
[0107] "FBS" means fetal bovine serum.
[0108] "Formulation(s)" means a combination of at least one active
ingredient with one or more other ingredients for one or more
particular uses, such as storage, further processing, sale, and/or
administration to a subject, such as, for example, administration
to a subject of a specific agent in a specific amount, by a
specific route, to treat a specific disease.
[0109] "Fragment(s)" herein means part of a larger entity, such as
a part of a protein; for instance, a polypeptide consisting of less
than the entire amino acid sequence of a larger polypeptide. As
used herein, the term includes fragments formed by terminal
deletion and fragments formed by internal deletion, including those
in which two or more non-contiguous portions of a polypeptide are
joined together to form a smaller polypeptide, which is a fragment
of the original.
[0110] "Fusion protein(s)" herein means a protein formed by fusing
all or part of two polypeptides, which may be either the same or
different. Typical fusion proteins are made by recombinant DNA
techniques, by end to end joining of nucleotides encoding the two
(or more) polypeptides.
[0111] "Genetically engineered" herein means produced using a
deliberate process of genetic alteration, such as by recombinant
DNA technology, classical methods of genetic manipulation, chemical
methods, a combination of all three, or other methods.
[0112] "Homolog(s)" herein means having homology to another entity,
such as a protein that is homologous to another protein. Homologous
means resembling in structure or in function.
[0113] "Ionization" herein means the change of net charge on a
substance by at least one, including loss or gain of charge, such
as the ionization of acetic acid in low pH solution, from HOAc to
OAc.sup.- and H.sup.+.
[0114] "k" herein denotes an equilibrium co-efficient, in
accordance with its standard meaning in chemistry.
[0115] "k.sub.a" herein denotes the dissociation constant of a
particular hydrogen of a molecule, in accordance with its standard
meaning in chemistry, such as, for example, the dissociation
constant of the acidic hydrogen of acetic acid.
[0116] "k.sub.d" herein denotes a dissociation constant of a pair
of chemical entities (or moieties), in accordance with its standard
meaning in chemistry.
[0117] "Kit" means a collection of items used together for a given
purpose or purposes.
[0118] "Ligand(s)" herein means a molecular entity that binds
selectively and stoichiometrically to one or more specific sites on
one more other molecular entities. Binding typically is
non-covalent, but can be covalent as well. A very few examples,
among many others, are (a) antigens, which typically bind
non-covalently to the binding sites on cognate antibodies; (b)
hormones, which typically bind hormone receptors, non-covalently;
(c) lectins, which bind specific sugars, non-covalently; (d)
biotins, which bind multiple sites on avidin and other avidin-like
proteins, non-covalently; (e) hormone antagonists, which bind
hormone receptors and inhibit their activity and/or that of the
corresponding hormone; and (f) hormone agonists, which similarly
bind hormone receptors but stimulate their activity.
[0119] "Ligand-binding moiety(s)" herein means a molecular entity
that binds a ligand, typically, a part of a larger molecular entity
that binds the ligand, or a molecular entity derived therefrom.
[0120] "Ligand-binding protein(s)" herein means a protein that
binds a ligand.
[0121] "Ligand moiety(s)" herein means a molecular entity that
binds to a ligand-binding molecular entity in much the same way as
does the corresponding ligand. A ligand moiety can be all of a
ligand, or part of it, derived from a ligand, or generated de novo.
Typically, however, the ligand moiety is more or less exclusively
the aspect thereof that binds corresponding ligand-binding
entities. The ligand moiety need not comprise, and the term
generally does not denote, structural features other than those
required for ligand binding.
[0122] "mEq" herein means milliequivalent(s).
[0123] ".mu.Eq" herein means microequivalent(s).
[0124] "Mimetic(s)" herein means a chemical entity with structural
or functional characteristics of another, generally unrelated
chemical entity. For instance, one kind of hormone mimetic is a
non-peptide organic molecule that binds to the corresponding
receptor in the same way as the corresponding hormone.
[0125] "mM" means millimolar; 10.sup.-3 moles per liter.
[0126] "Modified protein(s)," "modified polypeptide(s)," or
"modified fragment(s)" herein means a protein or a polypeptide or a
fragment of a protein or polypeptide comprising a chemical moiety
(structure) other than those of the twenty naturally occurring
amino acids that form naturally occurring proteins. Modifications
most often are covalently attached, but can also be attached
non-covalently to a protein or other polypeptide, such as a
fragment of a protein.
[0127] "Moiety(s)" herein means a molecular entity that embodies a
specific structure and/or function, without extraneous components.
For instance, in most cases, only a small part of a ligand-binding
protein is responsible for ligand binding. This part of the
protein, whether continuously encoded or discontinuously, is an
example of a ligand-binding moiety.
[0128] "Naturally occurring" means occurs in nature, without human
intervention.
[0129] "Non-naturally occurring" means does not occur in nature or,
if it occurs in nature, is not in its naturally occurring state,
environment, circumstances, or the like.
[0130] "PBS" means phosphate buffered saline.
[0131] "Peptibody" refers to a molecule comprising an antibody Fc
domain (i.e., C.sub.H2 and C.sub.H3 antibody domains) that excludes
antibody C.sub.H1, CL, V.sub.H, and V.sub.L domains as well as Fab
and F(ab)2, wherein the Fc domain is attached to one or more
peptides, preferably a pharmacologically active peptide,
particularly preferably a randomly generated pharmacologically
active peptide. The production of peptibodies is generally
described in PCT publication WO00/24782, published May 4, 2000,
which is herein incorporated by reference in its entirety,
particularly as to the structure, synthesis, properties, and uses
of peptibodies.
[0132] "Peptide(s)" herein means the same as polypeptide; often,
but not necessarily, it is used in reference to a relatively short
polypeptide,
[0133] "pH" is used in accordance with its well-known and universal
definition as follows:
pH=-log [H.sub.3O.sup.+].
[0134] "Pharmaceutical" as used herein means is acceptable for use
in a human or non-human subject for the treatment thereof,
particularly for use in humans, and approved therefor by a
regulatory authority empowered to regulate the use thereof such as,
for example, the Food and Drug Administration in the United States,
European Agency for the Evaluation of Medicinal Products, Japan's
Ministry of Health, Labor and Welfare, or other regulatory agency
such as those listed in R. Ng, DRUGS: FROM DISCOVERY TO APPROVAL,
Wiley-Liss (Hoboken, N.J.) (2004), which is herein incorporated by
reference in its entirety, particularly as to regulatory
authorities concerned with drug approval, especially as listed in
Chapter 7. As used herein the phrase "wherein the composition has
been approved for pharmaceutical use by an authority legally
empowered to grant such approval" means an entity or institution or
the like, established by law and by law charged with the
responsibility and power to regulate and approve the use of drugs
for use in humans, and in some cases, in non-humans. Approval by
any one such agency anywhere meets this qualification. It is not
necessary for the approving agency to be that of the state in
witch, for instance, infringement is occurring. Example of such
entities include the U.S Food and Drug Administration and the other
agencies listed herein above.
[0135] As used herein, "pharmaceutical" also may refer to a product
produced in accordance with good manufacturing practices, such as
those described in, among others, Chapter 9 and Chapter 10, of R.
Ng, DRUGS: FROM DISCOVERY TO APPROVAL, Wiley-Liss (Hoboken, N.J.)
(2004), which is herein incorporated by reference in its entirety,
particularly in parts pertinent to good manufacturing practices for
pharmaceutical protein formulations, in particular, as set forth in
Chapters 9 and 10.
[0136] "Pharmaceutically acceptable" is used herein in accordance
with its well-known meaning in the art to denote that which is
acceptable for medical or veterinary use, preferably for medical
use in humans, particularly approved for such use by the US Food
and Drug Administration or other authority as described above
regarding the meaning of "pharmaceutical."
[0137] "Polypeptide(s)" see "Protein(s)."
[0138] "Precursor(s)" is used herein in accordance with its
well-known meaning in the art to denote an entity from which
another entity is derived. For instance, a precursor protein is a
protein that undergoes processing, such as proteolytic cleavage or
modification, thereby giving rise to another precursor protein
(which will undergo further processing) or a mature protein.
[0139] "Protein(s)" herein means a polypeptide or a complex of
polypeptides, in accordance with its well-known meaning in the art.
As used herein, "protein(s)" includes both straight chain and
branched polypeptides. It includes unmodified and modified
polypeptides, including naturally occurring modifications and those
that do not occur naturally. Such modifications include chemical
modifications of the termini, the peptide backbone, and the amino
acid side chains; amino acid substitutions, deletions and
additions; and incorporation of unusual amino acids and other
moieties, to name just a few such modifications. It also includes
"engineered" polypeptides and complexes thereof, such as, but not
limited to, any polypeptide or complex of polypeptides that has
been deliberatively altered in its structure by, for instance,
recombinant DNA techniques, chemical synthesis, and/or covalent
modification, including deliberate alteration of amino acid
sequence and/or posttranslational modifications.
[0140] "Protonation" means the addition of at least one
hydrogen.
[0141] "Self-buffering" means the capacity of a substance, such as
a pharmaceutical protein, to resist change in pH sufficient for a
given application, in the absence of other buffers.
[0142] "Semi-de novo" herein means (a) partly designed in
accordance with a particular reference and or produced from a
precursor, and (b) partly designed without reference to a
particular reference (such as designed solely by general principles
and not based on any particular reference). For example, a
polypeptide made by producing a first peptide in a bacterial
expression system, producing a second peptide by chemical
synthesis, and then joining the two peptides together to form the
polypeptide.
[0143] "Semi-synthesis" means as used herein a combination of
chemical and non-chemical methods of synthesis.
[0144] "Subject" means a vertebrate, such as a mammal, such as a
human. Mammals include, but are not limited to, humans, farm
animals, sport animals, and pets. Subjects in need of treatment by
methods and/or compositions of the present invention include those
suffering from a disorder, dysfunction, or disease, or a side
effect thereof, or from a side effect of a treatment thereof.
[0145] "Substantially" is used herein in accordance with its plain
and ordinary definition to mean to a great extent or degree. For
example, substantially complete means complete to a great extent,
complete to a great degree. By way of further illustration,
substantially free of residue means to a great extent free of
residue, free of residue to a great degree. Should numerical
accuracy be required, depending on context, "substantially," as
used herein means, at least, 80% or more, particularly 90% or more,
very particularly 95% or more.
[0146] "Therapeutically effective" is used herein in accordance
with its well-known meaning in the art to denote that which
achieves an improvement in the prognosis or condition of a subject
or that otherwise achieves a therapeutic objective, including, for
instance, a reduction in the rate of progress of a disease even if
a subject's condition, nonetheless, continues to deteriorate.
[0147] "Therapeutically effective amount" generally is used to
qualify the amount of an agent to encompass those amounts that
achieve an improvement in disorder severity. For example, effective
neoplastic therapeutic agents prolong the survivability of the
subject, inhibit the rapidly-proliferating cell growth associated
with the neoplasm, or effect a regression of the neoplasm.
Treatments that are therapeutically effective within the meaning of
the term as used herein, include treatments that improve a
subject's quality of life even if they do not improve the disease
outcome per se.
[0148] "Treat," "treating," or "treatment" are used broadly in
relation to the invention and each such term encompasses, among
others, preventing, ameliorating, inhibiting, or curing a
deficiency, dysfunction, disease, or other deleterious process,
including those that interfere with and/or result from a
therapy.
[0149] "Variant(s)" herein means a naturally occurring or synthetic
version of, for instance, a protein that is structurally different
from the original but related in structure and/or function, such as
an allelic variant, a paralog, or a homolog of a protein.
DESCRIPTION OF THE INVENTION
[0150] The invention provides for the first time self-buffering
protein formulations, particularly biopharmaceutical protein
formulations, methods for making the formulations, and methods for
using the formulations, among other things. Any protein that
provides sufficient buffer capacity within the required pH range at
a concentration suitable for its intended use can be prepared as a
self-buffering protein formulation in accordance with the
invention. The invention can be practiced with a variety of
proteins, including both naturally-occurring proteins and
"engineered" proteins, particularly biopharmaceutical proteins, as
discussed further below.
[0151] The utility of proteins, particularly biopharmaceutical
proteins, to be formulated in self-buffering compositions,
particularly pharmaceutically acceptable compositions, has not been
recognized prior to the invention herein disclosed. The influence
of proteins in the regulation of physiological pH has been
recognized and studied for some time. However, it has not
heretofore been recognized that proteins, particularly
biopharmaceutical proteins, can have enough buffer capacity to
maintain a formulation within a desired pH range, without
additional buffering agents.
[0152] Biopharmaceutical proteins for use in the United States are
formulated as buffered solutions, unbuffered solutions, amorphous
or crystalline suspensions, and lyophilates.
[0153] Most of the buffered solution formulations use a
conventional buffering agent. Two proteins, Pulmozyme.RTM. and
Humulin.RTM., are formulated as solutions without conventional
buffering agents. Neither of these proteins provides substantial
self-buffering capacity in the formulations.
[0154] Pulmozyme.RTM. has a molecular weight of about 37,000
Daltons and contains 5 histidines, 22 aspartic acids, and 12
glutamic acids, among its 260 amino acids. The buffering capacity
of the protein within 0.5 pH units of pH 6.3 is determined
substantially by its histidine content. On this basis, the upper
limit of the self-buffering capacity of the formulation is
determined by the effective concentration of the histidine
residues, 0.15 mM. The molar concentration of aspartic acid and
glutamic acid in the formation is 0.9 mM. The total molar
concentration of all three amino acids together, thus, is just a
little over 1 mM, at the concentration of the formulation.
[0155] Humulin.RTM. is formulated at 3.5 g/ml. It has a molecular
weight of about 6,000 Daltons and contains 2 aspartic acids, 8
glutamic acids, and 2 histidines. None of these amino acids is a
particularly effective buffer at the pH of the formulation: 7.0 to
7.8. At this concentration the molar concentration of histidines,
which are closest in pK.sub.a to the pH of the formulation, is 1.16
mM.
[0156] The biopharmaceutical lyophilates are reconstituted prior to
use forming solutions or suspensions. Most of the lyophilates
contain conventional buffers that maintain the proper pH of the
reconstituted formulations. A few others, in which the protein
concentration is low or the pH must be low (less than 3) or high
(greater than 9.5), are, effectively unbuffered.
[0157] Thus, buffering is achieved in current biopharmaceutical
protein formulations using conventional buffering agents. The
ability of proteins by themselves to buffer pharmaceutical protein
formulations has not been fully appreciated and has not been used
for the manufacture of protein pharmaceuticals.
[0158] The determination of protein buffer capacity, typically, is
important to developing self-buffering protein formulations in
accordance with the invention. Pertinent thereto, methods for
measuring buffer capacity and for determining the buffer capacity
of proteins are described below. To allow ready comparability of
data, protein buffer capacity must be expressed in comparable units
and/or related to a buffer standard. Accordingly, the following
section describes pH metrics and standards that meet these
requirements, in accordance with the invention.
1. Buffering
[0159] A widely accepted definition of buffering is the resistance
to change in pH of a composition upon addition of acid or base.
Buffer capacity thus often is defined as the ability of a
composition to resist pH change.
[0160] Typically buffer capacity is expressed in terms of the
amount of strong acid or base required to change the pH of a
composition a given amount. Van Slyke provided the most widely used
quantitative measure of buffer capacity, according to which, for a
solution, buffer capacity is expressed as the amount of strong acid
or base required to change the pH of one liter of the solution by
one pH unit under standard conditions of temperature and
pressure.
[0161] According to this measure, for instance, the buffer capacity
of 1 liter of 5 mM HOAc, 5 mM NaOAc, pH 4.76 in pure water is
4.09.times.10.sup.-3 moles of a univalent strong base (i.e.,
4.09.times.10.sup.-3 equivalents of base), which can be calculated
as follows.
[0162] The Henderson-Hasselbalch equation for the solution is:
pH=log {[5 mM]NaOAc/[5 mM]HOAc}+4.76
[0163] Accordingly, the concentration, X, of a univalent strong
base required to increase the pH of this buffer is:
4.76 to 5.76 is 5.76=log {[5 mM+X mM]NaOAc/[5 mM-X
mM]HOAc}+4.76
[0164] Thus:
1.00=log {[5 mM+XmM]NaOAc/[5 mM-X mM]HOAc}
10.0=[5 mM+X mM]NaOAc/[5 mM-X mM]HOAc
10.0=(5 mM+X m)/(5 mM-X mM)
50 mM-10X mM=5 mM+X mM
11X mM=45 mM
X=4.09 mM,
which, for one liter yields:
(4.09.times.10.sup.-3 moles/liter)(1 liter)(1
equivalent/mole)=4.09.times.10.sup.-3 equivalents
[0165] Thus, according to this measure, the buffer capacity of 1
liter of a 10 mM acetate buffer containing 5 mM NaOAc and 5 mM HOAC
at a pH of 4.76 in pure water is 4.09.times.10.sup.-3 equivalents
of base per liter per pH unit. Put other ways, the buffer capacity
of the solution is 4.09 milliequivalents of base per liter per pH
unit, 4.09 microequivalents of base per milliliter per pH unit,
0.409 microequivalents of base per 100 microliters per pH unit,
40.9 nanomoles of base per 10 microliters per pH unit, and 4.09
nanomoles of base per microliter per pH unit.
[0166] The same calculation yields the following buffer capacity
for other concentrations of this acetate buffer at pH 4.76. A 2 mM
acetate buffer as above has a buffer capacity of 0.818 mEq per
liter per pH unit. At 4 mM the buffer capacity is 1.636 mEq per
liter per pH unit. The capacity at 5 mM is 2.045 mEq per liter per
pH unit. At 7.5 mM the capacity is 3.068 mEq per liter per pH unit.
At 10 mM the acetate buffer has a buffer capacity of 4.091 mEq per
liter per pH unit. At 15 mM its capacity is 6.136 mEq per liter per
pH unit.
[0167] It is worth noting that an acetate buffer solution at the
pK.sub.a of acetic acid (pH 4.76) is equimolar in acetic acid and
acetate base. (i.e., at the pK.sub.a the acid and base are present
in equal amounts). As a result, the resistance to change in pH
(buffer capacity) of an acetate buffer at the pK.sub.a of acetic
acid is the same for addition of acid and base. The equipoise to
acid and base is a general characteristic of buffering agents in
buffers at a pH equal to their pK.sub.a.
[0168] At any other pH a buffer will contain different amounts of
acid and base forms and, therefore, its resistance to change (i.e.,
its buffer capacity) upon addition of acid will not be the same as
its resistance to change upon addition of base. As a result, it is
preferable to define the capacity of such buffers in terms of (i)
the amount of acid required to lower the pH by one unit, and (ii)
the amount of base required to raise the pH by one unit.
[0169] The partitioning in a buffer between acid and base forms in
a given composition, such as a pH standard, can be calculated at
any pH and buffer concentration using the procedures set forth
above in describing the buffer capacity of 10 mM NaOAc at pH 4.76
plus or minus (containing equimolar amounts of acetic acid and
sodium acetate). And the results can be used to define the buffer
capacity of a standard for reference use.
[0170] Thus, for instance, the partitioning of acetic acid into
acetic acid and acetate base in a solution at pH 5.0 can be
calculated readily using the foregoing procedures, and from this
the buffer capacity can be calculated for both base and for acid
addition. Calculated this way, the theoretical buffer capacity of
10 mM sodium acetate buffer over the range from pH 5.0 to 5.5 is
approximately 2.1 mM per 0.5 pH unit and 4.2 mM per pH unit. Put
another way, the buffer capacity of the buffer, theoretically, is
approximately 4.2 .mu.Eq per ml of buffer solution per unit of pH
change. Similarly, the theoretical buffer capacity of 10 mM sodium
acetate buffer over the range from pH 5.0 to 4.0 is 4.9 mM, and,
put another way, 4.9 .mu.Eq per ml of buffer per unit of pH change
over a given range of pH.
[0171] While such calculations often are quite useful in many
cases, empirical standards and empirical determinations are
preferred. Among particularly preferred empirical standards are
sodium acetate buffers over the range of pH 5.0 to 4.0 and pH 5.0
to 5.5 as exemplified in Examples 1 and 2. Especially preferred are
sodium acetate buffers in accordance therewith in which the total
acetate concentration is, in particular, 10 mM, preferably 5 mM,
especially 4 mM, among others as set forth elsewhere herein.
[0172] Acetate buffers at pH 5.0 are more resistant to change in pH
upon addition of acid than upon addition of base, as discussed
above. In a preferred empirical standard of buffer capacity, the
buffer capacity of a standard acetate buffer such as these is
defined as: (i) the slope of the least squares regression line
calculated for base titration data for the buffer from pH 5.0 to pH
5.5, and (ii) the slope of the least squares regression line
calculated for acid titration data for the buffer from pH 5.0 to pH
4.0. The preparation of standard acetate buffers and the
determination of their buffer capacities are described in Examples
1, 2, and 3. It is to be appreciated that much the same methods can
be used to establish and use buffer capacity standards using other
suitable buffering agents.
[0173] In measuring the buffer capacity of a self-buffering protein
composition in accordance with the invention, it often is
convenient to express the buffer capacity in terms of the
concentration of a standard buffer at the same pH having the same
buffer capacity. When a standard is used that is not at the
pK.sub.a of the buffering agent, such as a sodium acetate buffer
initially at pH 5.0, in accordance with the invention the
self-buffering composition is defined as having a buffer capacity
equal to or greater than that of the standard, if either its buffer
capacity upon base titration or its buffer capacity upon acid
titration (or both) is equal to or exceeds the corresponding buffer
capacity of the standard.
[0174] It is to be further appreciated that the pH of
self-buffering protein compositions in accordance with the
invention generally will not be at the pK.sub.a of the
self-buffering protein, or any acid-base substituent therein.
Indeed proteins are polyprotic and, as discussed herein, often will
have several substituents, each with a somewhat different pH that
contribute to its buffer capacity in a given pH range. Accordingly,
the buffer capacity of self-buffering protein formulations in
accordance with the invention preferably is determined empirically
by both acid titration and base titration over a given range of pH
change from the desired pH of the composition. In preferred
embodiments in this regard, the buffer capacity is determined by
titrating with acid and separately with base over a change of
respectively + and -1 pH unit from the starting pH of the
formulation. In particularly preferred embodiments, the titration
data is collected for a change in pH of plus or minus 0.5 pH units.
As described in the Examples, the buffer capacity is the slope of
the least squares regression line for the data for pH as a function
of equivalents of acid or base added to the composition over the
range of titration.
[0175] a. Empirical Measures and Standards of Buffer Capacity
[0176] In certain preferred embodiments of the invention, the
measure of buffer capacity is an empirical standard. Among
preferred empirical standards in this regard are a particular
volume of an aqueous solution at a particular temperature and a
particular pH, containing a particular buffering agent at a
particular concentration and either no other components than water,
or one or more other particular components, each at a particular
concentration.
[0177] A particularly preferred specific standard for determining
buffer capacity in accordance with various aspects and preferred
embodiments of the invention is 10 mM sodium acetate pH 5.00 in
pure water free of other constituents at 21.degree. C. in
equilibrium with ambient air at 1 atmosphere, as described in
Examples 1 and 2, preferably expressed in equivalents per unit
volume per pH unit, such as .mu.Eq/ml-pH. Buffer capacity of the
standard should be measured empirically as described in Examples 1,
2, and 3, and as further discussed elsewhere herein.
[0178] A particularly preferred specific standard for determining
buffer capacity in accordance with various aspects and preferred
embodiments of the invention is 10 mM sodium acetate pH 4.76 in
pure water free of other constituents at 21.degree. C. in
equilibrium with ambient air at 1 atmosphere, as described in
Examples 1 and 2, preferably expressed in equivalents per unit
volume per pH unit, such as .mu.Eq/ml-pH. Buffer capacity of the
standard should be measured empirically as described in Examples 1,
2, and 3, and as further discussed elsewhere herein. According to
the Henderson-Hasselbalch equation, as noted above, the calculated
buffer capacity of this standard over the range of pH 4.76 plus or
minus 1 pH unit is 4.09 microequivalents per milliliter per pH unit
(4.09 .mu.Eq/ml-pH).
[0179] A variety of other buffers are available for use as
standards in other ranges of pH in accordance with various aspects
and preferred embodiments of the invention in this regard.
Reference buffers are particularly preferred in this regard, such
as those well-known and routinely employed for analytical chemistry
determinations. A variety of such buffering agents are set forth in
textbooks on analytical chemistry and in monographs on the accurate
determination of pH and buffer capacity.
[0180] Also useful in the invention in this regard are biological
buffers, such as those described in, among other texts: TEITZ
TEXTBOOK OF CLINICAL CHEMISTRY, 3.sup.rd Ed., Burtis and Ashwood,
eds., W.B. Saunders Company, Philadelphia, Pa. (1999), in
particular in Tables 50-13 to 50-16, which are herein incorporated
by reference in their entireties as to buffering agents and buffers
and their use as pH and/or buffer capacity standards in accordance
with the invention in this respect; THE TOOLS OF BIOCHEMISTRY,
Terrance G. Cooper, John Wiley & Sons, New York, N.Y. (1977),
in particular Chapter 1, pages 1-35, which is herein incorporated
by reference in its entirety as to buffering agents and buffers and
their use as pH and buffer capacity standards in accordance with
the invention in this respect, most particularly as to Tables 1-3,
1-4, and 1-5 and text relating thereto, and PROTEIN PURIFICATION
PRINCIPLES AND PRACTICE, 3.sup.rd Ed., Robert K. Scopes,
Springer-Verlag, New York, N.Y. (1994), in particular pages
160-164, especially therein Tables 6.4 and 6.5 and text relating
thereto, Chapter 12, section 3, pages 324-333, especially therein
Tables 12-4 and 12-5 and text relating thereto, and all of Appendix
C: Buffers for Use in Protein Chemistry, which are herein
incorporated by reference in their entirety as to buffering agents
and buffers and their use in accordance with the invention in this
respect.
[0181] Since some dissolved gases in water react with OH.sup.-
and/or H.sub.3O.sup.+, however, the empirically determined buffer
capacity of the standard solution may vary somewhat from the
theoretical value. Hence, the definition of the standard requires
that the solution be in equilibrium with the atmosphere at a
pressure of 1 atmosphere. In addition, the buffer standard must be
held in and contacted only with materials that do not alter its
components or its buffer capacity, such as those that leach acids,
bases, or other reactants that may alter the effective
concentration or activity of the acetate buffer in any way that
would alter its buffer capacity. Given both of the foregoing,
atmospheric equilibration and inertness of the container, buffer
capacity of the standard will scale directly and linearly with its
volume. Accordingly, the buffer capacity of 100 ml will be 1/10
that of 1.00 liter, and the buffer capacity of 10 ml will be 1/100
that of 1.00 liter. Accordingly, the volume of the standard can be
adjusted for convenience and then normalized back to 1 liter as
desired.
[0182] It may not always be convenient to make the foregoing 10 mM
acetate buffer capacity standard for field use. However, a variety
of other buffer capacity standards can be made and used in the same
way as the acetate buffer, using a variety of other buffering
agents. Provided only that the buffering standards are prepared
properly, they can be calibrated against the acetate buffering
standard described above and then used in the field. The results
obtained with such alternative standards may then be expressed in
terms of the foregoing acetate standard without substantial
distortion or error.
[0183] The buffer capacity of such alternative standards also can
be calibrated by calculation. To do so, the buffer capacity of the
alternative standard is determined directly and expressed in mEq
per unit volume per unit of pH. Determinations based on the
alternative standard then can be normalized to the acetate standard
using the ratio between the buffering capacities expressed in mEq
per unit volume per unit of pH of the alternative and the acetate
standards.
[0184] Using such methods, which are commonly employed in metrology
to relate field standards back to a reference standard, the acetate
buffer standard described above provides a portable, scalable,
reliable, and accurate reference for determining the buffer
capacity of any composition that readily can be compared with
disparate measures made on other compositions using similar
methods.
[0185] b. Preparation of Buffer Capacity Standards
[0186] Buffer capacity standards can be prepared using
well-established methods of analytical chemistry. See for instance,
ANALYTICAL CHEMISTRY, 3.sup.rd Ed., Douglas A. Skoog and Donald M.
West, Holt, Rinehart and Winston, New York (1979), particularly
chapter 9 (pages 186-226), chapter 10 (pages 227-233), and methods
described on pages 583-588; TEITZ TEXTBOOK OF CLINICAL CHEMISTRY,
3.sup.rd Ed., Burtis and Ashwood, eds., W.B. Saunders Company,
Philadelphia, Pa. (1999), in particular Chapter 1 regarding general
laboratory techniques for preparing and calibrating buffers and
Tables 50-13 to 50-16; THE TOOLS OF BIOCHEMISTRY, Terrance G.
Cooper, John Wiley & Sons, New York, N.Y. (1977), in particular
Chapter 1, pages 1-35, and Tables 1-3, 1-4, and 1-5 and text
relating thereto; PROTEIN PURIFICATION PRINCIPLES AND PRACTICE,
3.sup.rd Ed., Robert K. Scopes, Springer-Verlag, New York, N.Y.
(1994), in particular pages 160-164, especially therein Tables 6.4
and 6.5 and text relating thereto, Chapter 12, section 3, pages
324-333, especially therein Tables 12-4 and 12-5 and text relating
thereto, and all of Appendix C: Buffers for Use in Protein
Chemistry; and REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY,
21.sup.st Ed., Beringer et al. Editors, Lippincott, Williams &
Wilkins, Philadelphia, Pa. (2005), particularly in parts relating
to buffering agents, buffers, buffer capacity and the like; each of
which is herein incorporated by reference in its entirety
particularly as to the preparation and use of buffers and buffer
capacity standards in accordance with the invention in this
respect.
[0187] The water used for preparing buffer capacity standards
should be highly purified, preferably Type I water, such as milliQ
water, or triple distilled water. The buffer reagents should be
pure and, in particular, free of any substance that can alter the
pH or buffer capacity of the standard solution, such as Reference
Grade or ACS Reagent Grade reagents suitable for use in demanding
analytic chemical analyses, as described in the foregoing
references, TEITZ and REMINGTON cited above in particular, which
are hereby incorporated by reference in their entireties
particularly in parts pertinent to analytical grade water and
reagents.
[0188] The exact compositions of the buffer reagents must be well
established. The molecular weight of the buffer reagents must be
known accurately for each buffer reagent. The molecular weights
must be for the exact reagent that will be used and must include
the weight of adducts such as hydrates that are present in the
reagent. The effective number of hydrogen donors or hydrogen
acceptors per molecule must be known accurately for each buffer
reagent. The proportional distribution of different forms, such as
hydrates, must be known for each reagent that contains a mixture of
such forms. Concentrations of liquid buffer reagents much be known
exactly, preferably in moles/volume and in moles/mass (e.g.,
moles/liter and moles/gm or kg. Hygroscopic agents must be dried to
remove moisture so that reagent can be accurately weighed.
[0189] Generally speaking, the information provided by
well-established vendors of reagents and reference grade chemicals
is sufficiently accurate for the preparation of buffer capacity
standards as described above. And well-known standard techniques
routinely employed in analytical chemistry can be used to dry
"hygroscopic reagents" so that they can be weighed accurately.
[0190] As described therein, well established and routinely
employed analytical chemistry methods can be employed to prepare
and calibrate acid and base solutions, such as 1 N HCl and 1 N NaOH
(to name just two) for titrating buffer capacity standard
solutions, as well as sample protein solutions, to determine buffer
capacity. It should be noted that the preparation of NaOH solutions
for titration should be done so as to eliminate inaccuracies that
arise from the interaction of certain dissolved gases with basic
solutions, and the pH altering effects of their salvation. See for
instance Skoog and West (1979) and other references cited above
regarding the preparation and calibration of buffers and buffer
standards, which are herein incorporated by reference in their
entireties particularly in parts pertinent to the preparation of
standard solutions for titration, as discussed above.
[0191] c. Empirical Measurement of Buffer Capacity
[0192] Titration of standards and samples to determine buffer
capacity can be done using well-known, routine methods. Titrations
can be carried out manually. They also can be carried out using an
autotitrator. A wide variety of autotitrators that are suitable for
use in the invention in this regard are commercially available from
numerous vendors. Methods suitable for use in the invention in this
regard are the same as those described in the references cited
above regarding preparation and calibration of buffer standards,
each of which is incorporated herein by reference in its entirety
particularly in parts pertinent to the titration of known and
unknown solutions to determine their buffer capacity.
2. Buffering by Proteins and Protein Buffer Capacity
[0193] a. Determination of Protein Hydrogen Equilibria and Buffer
Capacity
[0194] Proteins invariably contain many acidic and basic
constituents. As a result hydrogen ion equilibrium of proteins is
highly complex. In fact, a complete description of the hydrogen ion
equilibria of a protein in a given environment is beyond the reach
of current theoretical and computational methods. Empirical
measurements of protein buffer capacities, thus are preferred.
Methods developed for precise empirical measurement of protein
hydrogen equilibria, which can be and are routinely employed by
those skilled in the art, are well-suited to measuring the
buffering properties of proteins pertinent to the development of
self-buffering protein formulations in accordance with the
invention. Thus, the pH titration curves of proteins can be
determined in accordance with the invention by well-known methods
such as those described in and exemplified by pH titration studies
of Tanford and co-workers on ribonuclease. See C. Tanford,
"Hydrogen Ion Titration Curves of Proteins," in T. Shedlovsky
(ed.), ELECTROCHEMISTRY IN BIOLOGY AND MEDICINE, John Wiley and
Sons, New York, 1955, Ch. 13; C. Tanford and J. D. Hauenstein, J.
Am. Chem. Soc. 78, 5287 (1956), C. Tanford, PHYSICAL CHEMISTRY OF
MACROMOLECULES, John Wiley and Sons, New York, 1961, particularly
pages 554-567, all of which are herein incorporated by reference
particularly in parts pertinent to hydrogen ion titration of
proteins and to the determination of buffering action and buffer
capacity of proteins.
[0195] However, the present invention does not require such precise
determinations as those described in the foregoing references.
Rather, the buffering properties and buffer capacity of proteins in
accordance with the invention can be determined using the methods
described in standard references on analytical chemistry and
biochemistry, such as, for instance, Skoog (1979), Cooper (1977),
and Scopes (1994), cited above, each of which is herein
incorporated by reference in its entirety particularly as to the
empirical determination of titration curves, particularly of
proteins within a given range of pH in accordance with the
invention.
[0196] The determination of titration curves and buffer capacity in
accordance with the invention is described in detail for numerous
acetate buffers and a variety of pharmaceutical proteins in the
Examples below. Thus, the pH titration curves of proteins can be
determined empirically in accordance with such methods as described
in the foregoing references over particular limited ranges of pH
that are of interest to a given formulation. In many respects these
methods are the same as those used in analytical chemistry for the
titration of small molecules such as acetate buffers (as
illustrated in the Examples). Somewhat greater care must be taken,
however, in handling proteins to maintain the conformation and
function required for effective formulation.
[0197] Protein titrations may be carried out manually or using
automated titrators. Equipment for manual titration and automated
titrators are readily available from a large number of suppliers
and vendors. Methods suitable for determining pH titration curves
and buffer capacity of proteins are exemplified in the Examples by
reference to titration of acetate buffer standards and to titration
of several different therapeutic proteins over defined ranges of
pH. These methods can be employed to determine the hydrogen
ionization behavior and buffer capacity of any other protein in
accordance with the invention.
[0198] It is a particular aspect of the invention to determine the
buffer capacity of proteins as a function of concentration in
solution. In a preferred method in this regard, solutions of a
given protein are prepared in a graded series of concentrations. A
pH titration curve is determined for the protein at each
concentration over the pH range of interest. Preferably titration
curves are determined for the range of interest using both base
titration and acid titration. The data are, in certain preferred
embodiments, plotted on a graph of equivalents of acid or base
added versus the measured pH of each solution. Typically, for
ranges of about 0.5 to 1.0 pH unit, the titration data for each
concentration closely fit a straight line, preferably determined by
a least squares regression analysis. In preferred embodiments in
this regard, buffer capacity for the protein at each concentration
is equated to the slope of the regression line, expressed in units
of equivalents per ml per pH unit (or fractions thereof). Also
useful in the invention in this regard is the relationship between
the buffer capacity of the protein and its concentration. In
certain preferred embodiments, this relationship is determined by a
least squares regression analysis of the best straight line fit of
the buffer capacity data determined in accordance with the
foregoing plotted on a graph of buffer capacity versus protein
concentration.
[0199] Empirical data on the buffer capacity of proteins in
accordance with the invention preferably is related to the buffer
capacity of a standard acetate buffer. That is, in particularly
preferred embodiments of the invention in this regard, the buffer
capacity of a given protein at a given concentration in a given
formulation, determined as above, is equated to the concentration
of a standard acetate buffer having the same buffer capacity.
[0200] While empirical determinations as described herein are
generally a crucial aspect of formulating self-buffering
compositions in accordance with various aspects and preferred
embodiments of the invention, theoretical and computational methods
also can be productively employed to guide the design, manufacture,
and use of such compositions (in conjunction with empirical
determinations), as described below.
[0201] b. Prediction of Protein Hydrogen Ion Equilibria and Buffer
Capacity
[0202] The ionization of hydrogen in proteins is complex but can be
broken down in general terms into pH ranges defined by the
ionizable hydrogens of amino acid side chains, and the terminal
amino and carboxyl groups. The pK.sub.a of terminal carboxyls in
polypeptides typically ranges around 3.1. The pK.sub.a of the
acidic hydrogens in the side chains of aspartic acid and glutamic
acid range around 4.4. The pK.sub.a of histidine in polypeptides
ranges around 6.0. The terminal amino group hydrogen ionization
pK.sub.a typically ranges around 7.5. The sulfhydryl in cysteine
has a pK.sub.a range around 8.5. The tyrosine hydroxyl and the
lysine amine both have pK.sub.as ranging around 10. The pK.sub.a of
arginine ranges around 12.
[0203] Conformational folding typically partitions large
polypeptides and proteins in polar solvents into exposed
solvent-accessible regions and more or less non-polar core regions
that have little or no contact with the ambient environment.
Folding produces many environments between these two extremes.
Furthermore, the micro environment around a given amino acid side
chain in a protein typically is affected by one or more of: solvent
effects; binding of ions; chelation; complexation; association with
co-factors; and post-translational modifications; to name just a
few possibilities. Each of these can influence the pK.sub.a of a
given amino acid ionization in a protein. The pK.sub.as for
specific residues in a given protein, thus, can vary dramatically
from that of a free amino acid.
[0204] Indeed, the perturbation of pK.sub.as by microenvironments
of amino acids in proteins has been used to study the folding of
proteins and the disposition and charge state of specific amino
acids in folded proteins. The protein titration curves reported by
Tanford and others are complex with a few broad features in common.
Typically only some of the ionizable protons are accounted for in
the titration curves. Others apparently are located in the core and
are inaccessible to solvent. The pK.sub.as of individual side
chains of the same type that can be detected in some cases can be
distinguished from one another. Nonetheless, while detectably
different, their pK.sub.as generally are close to that of the free
amino acid.
[0205] The strongest buffering action of proteins does not
generally occur at the isoelectric point, as may be mistakenly
supposed. In fact, buffering depends on the amino acid side chain
hydrogens and the terminal hydrogens, and therefore occurs in
ranges spanning the pK.sub.as of the ionizable hydrogens in the
free amino acids, as discussed above. The most important of these,
for formulating compositions of proteins, especially certain
pharmaceutical proteins that are more soluble and/or more stable,
among other things, at weakly acidic pH (pH 4 to 6), is buffering
action that occurs in the range of the pK.sub.as of the carboxyl
hydrogen of the amino acids aspartic acid and glutamic acid; that
is, pH 4.0 to 5.5, particularly around 4.5.
[0206] There are a variety of ways available for estimating the
buffer capacity of a given protein in a given solution at a given
pH. Methods range from highly technical and complex computer models
to those that can be carried out on a hand calculator. None of the
methods is complete or entirely accurate; but, they can in some
instances provide useful estimates.
[0207] For instance, a potentially useful idea of buffer capacity
in some instances may be calculated for a protein in a solution
based on its amino acid composition, the pK.sub.as (in the solvent
in question) of the terminal amine and carboxy groups and the amino
acid side hydrogen donors and acceptors, the concentration of the
protein, and the pH of the solution.
[0208] For example, a potentially useful estimate of the buffer
capacity of a protein at pH in the range of the pK.sub.a of the
side chain carboxyl hydrogen of glutamic acid (as a free amino
acid), can be gained from the molecular weight of the protein and
the number of glutamic acid residues it contains. Dividing the
former by the latter provides the weight per equivalent of glutamic
acid and, therefore, the weight per equivalent of ionizable
hydrogen at the pK.sub.a of glutamic acid. Since glutamic acid and
aspartic acid side chain carboxyl groups have nearly the same
pK.sub.as, results of such calculations for the two should be added
together to yield an estimate of buffer capacity in a range around
both their pK.sub.as. The estimated buffer capacity of a solution
of the protein at the pK.sub.a can be calculated from the protein's
concentration in the solution and the intrinsic factor just
provided, namely weight per equivalent of ionizable hydrogen.
Dividing the concentration by the weight per equivalent yields an
estimate for the buffer capacity in units of Eq/volume. Such
estimates often will be too high, since some residues usually are
sequestered in regions of the protein not accessible to the
solvent, and, therefore, do not contribute to its actual buffer
capacity. It may be possible in certain instances to account for
the effect of sequestering on buffer capacity. For instance, a
fractional co-efficient that reflects theoretical or empirical
estimates of sequestering can be applied to adjust the original
calculation.
[0209] Such calculations generally will be of less utility and less
accurate than empirical determinations of protein buffer capacity,
in accordance with the methods described elsewhere herein. But they
can be useful to provide rough maximum estimates of the buffer
capacity of proteins in solution.
3. Proteins
[0210] The invention herein disclosed may be practiced with any
protein that provides sufficient buffer capacity in a desired pH
range within the parameters of protein concentration and the like
required for a desired formulation. Among preferred proteins in
this regard are pharmaceutical proteins for veterinary and/or human
therapeutic use, particularly proteins for human therapeutic use.
Also among preferred proteins are proteins that are soluble in
aqueous solutions, particularly those that are soluble at
relatively high concentrations and those that are stable for long
periods of time. Additionally, among preferred proteins are those
that have a relatively high number of solvent accessible amino
acids with side chain hydrogen ionization constants near the pH of
the desired buffering action.
[0211] Further among preferred proteins of the invention are
proteins for pharmaceutical formulations that do not induce a
highly deleterious antigenic response following administration to a
subject. Preferred in this regard are proteins for veterinary
and/or human medical use, particularly, regarding the latter,
humanized and human proteins.
[0212] Further among preferred proteins of the invention are
proteins that bind selectively to specific targets, including
ligand-binding proteins and protein ligands. Antigen-binding
proteins, proteins derived therefrom, and proteins related thereto
are among the particularly preferred embodiments of the invention
in this regard. Highly preferred proteins of the invention in this
regard are antibodies and proteins derived from antibodies or
incorporating antibodies, in whole or part, including, to name just
a few such entities: monoclonal antibodies, polyclonal antibodies,
genetically engineered antibodies, hybrid antibodies, bi-specific
antibodies, single chain antibodies, genetically altered
antibodies, including antibodies with one or more amino acid
substitutions, additions, and/or deletions (antibody muteins),
chimeric antibodies, antibody derivatives, antibody fragments,
which may be from any of the foregoing and also may be similarly
engineered or modified derivatives thereof, fusion proteins
comprising an antibody or a moiety derived from an antibody or from
an antibody fragment, which may be any of the foregoing or a
modification or derivative thereof, conjugates comprising an
antibody or a moiety derived from an antibody, including any of the
foregoing, or modifications or derivatives thereof, and chemically
modified antibodies, antibody fragments, antibody fusion proteins,
and the like, including all of the foregoing.
[0213] a. Antibodies, Antibody-Derived, and Antibody-Related
Proteins and the Like
[0214] Among particularly preferred proteins in accordance with the
invention are antibody polypeptides, such as heavy and light chain
polypeptides that have the same amino acid sequence as those that
occur in and make up naturally-occurring antibodies, such as those
that occur in sera and antisera, including such polypeptides and
proteins isolated from natural sources, as well as those that are
made by hybridoma technologies, by activation of an endogenous gene
(by homologous or non-homologous recombination, for instance), by
expression of an exogenous gene under the control of an endogenous
transcription control region, by expression of an exogenous
expression construct, by semi-synthesis and by de novo synthesis,
to name some techniques commonly employed for making antibodies and
antibody-related polypeptides and proteins that can be used to
produce antibody polypeptides and proteins in accordance with the
invention.
[0215] Included among these antibody-related polypeptides and
proteins are those in whole or part having a de novo amino acid
sequence, those that comprise all or one or more parts of an
antibody (that is: a continuous chain of amino acids having the
same sequence as any four or more residues in the amino acid
sequence of a naturally occurring antibody polypeptide), those
having an amino acid sequence that matches in some way that of a
naturally occurring antibody, but differs from it in other ways,
those that have the same but different amino acid sequences as a
naturally occurring counterpart or sequence relating thereto, but
differ from the counterpart in one or more post-translational
modifications, and those comprised in part of any of the foregoing
(in part or in whole) fused to one or more polypeptide regions that
can be of or derived from or related to a second, different
antibody polypeptide, and can be of or derived from any other
polypeptide or protein, whether naturally occurring, resembling but
differing therefrom, having a semi-de novo amino acid sequence
and/or a de novo sequence, among others. Such hybrids are generally
referred to herein as fusion polypeptides and/or fusion
proteins.
[0216] Further among preferred proteins in accordance with the
invention herein described are modified proteins in accordance with
all of the foregoing. Included among such modified proteins are
proteins modified chemically by a non-covalent bond, covalent bond,
or both a covalent and non-covalent bond. Also included are all of
the foregoing further comprising one or more post-translational
modifications which may be made by cellular modification systems or
modifications introduced ex vivo by enzymatic and/or chemical
methods, or introduced in other ways.
[0217] Among preferred proteins of the invention in this regard are
Fab fragment(s), such as those produced by cleaving a typical
dimeric (LH).sub.2 antibody with certain protease that leave the
light chain intact while cleaving the heavy chains between the
variable region and the adjacent constant region, "above" the
disulfide bonds that hold the heavy chains together. Such cleavage
releases one Fc fragment comprising the remaining portions of the
heavy chains linked together, and two dimeric Fab fragments each
comprising an intact light chain and the variable region of the
heavy chain. Fab fragments also can be produced by other techniques
that do not require isolation of a naturally occurring antibody
and/or cleavage with a protease.
[0218] Also preferred are Fab.sub.2 fragment(s) such as those
produced in much the same manner as Fab fragments using a protease
that cleaves "between or below" the disulfide bonds. As a result,
the two Fab fragments are held together by disulfide bonds and
released as a single Fab.sub.2 fragment. Fab.sub.2 fragments can be
produced by many other techniques including those that do not
require isolation of an intact antibody or cleavage with a protease
having the required specificity. Furthermore, both mono- and
bi-specific Fab.sub.2 fragments can now be made by a variety of
routine techniques.
[0219] Also among preferred proteins in this regard are Fab.sub.3
fragments, which are engineered antibody fragments in which three
Fab fragments are linked together. Fab.sub.3 fragments can be
mono-, bi-, or tri-specific. They can be made in a variety of ways
well-known to those of skill in the pertinent arts.
[0220] Among other preferred proteins in this regard are Fc
fragments(s), such as those produced by cleavage with a protease in
the same manner used for the production of either Fab fragments or
Fab.sub.2 fragments. However, for the production of Fc fragments,
the dimeric heavy chain containing fragments are isolated rather
than the light chain containing fragments. Fc fragments lack
antigen combining sites, but comprise effector regions that play a
role in physiological processes involving antibodies. Fc fragments
can be made by a variety of techniques that are well-known and
routinely employed by those of skill in the art for this
purpose.
[0221] Among other preferred proteins in this regard are
single-chain variable fragments ("scFv(s)"). scFv(s) are fusion
proteins made by joining the variable regions of the heavy and
light chains of an immunoglobulin. The heavy and light chains in an
scFv typically are joined by a short serine, glycine linker.
scFv(s) have the same specificity as the antibodies from which they
were derived. Originally produced through phage display, scFv(s)
now can be made by a variety of well-known methods.
[0222] Also preferred are Bis-scFv(s) which are fusions of two
scFv(s). Bis-scFv(s) can be mono- or bi-specific. A variety of
methods are well-known and can be applied in making Bis-scFv(s) in
accordance with the invention.
[0223] Also preferred in accordance with the invention in this
regard are minibodies; mono- and bi-specific diabodies; mono-, bi-,
and tri-specific triabodies; mono-, bi-, tri-, and tetra-specific
tetrabodies; VhH domains; V-NAR domains; V.sub.H domains; V.sub.L
domains; camel Igs; Ig NARs; and others.
[0224] Also among preferred embodiments in accordance with various
aspects and preferred embodiments of the invention in these and
other regards are proteins comprising one or more CDR and/or
CDR-derived and/or CDR-related regions of an antibody or one or
more FR and/or FR-derived and/or FR-related regions of an antibody.
In this regard CDR means complementary determining region; that is,
a hypervariable region of a light or heavy chain of an antibody,
typically about 9 to 12 amino acids in length that usually is an
important part of an antigen specific binding moiety of an
antibody. FR in this regard means a framework region of an
antibody; that is, a region of about 15 to 20 amino acids that
separates CDRs in the antigen specific binding moiety of an
antibody. The terms CDR-derived and CDR-related, and the terms
FR-derived and FR-related have the same meanings as to CDR and FR,
respectively, as set forth in the above Glossary for the terms
antibody-derived and antibody-related as to the term antibody.
[0225] Regarding antibodies, antibody-derived, and antibody-related
proteins in accordance with the foregoing and with other aspects of
the invention herein disclosed, see, for instance, Protein
Engineering: Principles and Practice, Jeffrey L. Cleland and Chares
S. Craik, eds. Wiley-Liss, Inc., New York (1996), particularly
therein Kelley, Robert F., "Engineering Therapeutic Antibodies,"
Chapter 15, pp. 399-434 and Hollinger, P. & Hudson, P.,
"Engineered antibody fragments and the rise of single domains,"
Nature Biotechnology, September 2005, 1126-1136, each of which is
herein incorporated by reference in its entirety particularly in
parts pertinent to the structure and engineering of antibodies,
particularly biopharmaceutical antibodies, and antibody-derived and
antibody-related proteins, particularly antibody-derived and
antibody-related pharmaceutical proteins in accordance with the
invention herein described.
[0226] As to all of the foregoing, particularly preferred in the
invention are human, humanized, and other proteins that do not
engender a significantly deleterious immune responses when
administered to a human. Also preferred in the invention are
proteins in accordance with all the foregoing that similarly do not
cause a significantly deleterious immune responses on
administration to non-humans.
[0227] Among very particularly preferred proteins in accordance
with the invention in these regards are fusion proteins comprising
antibodies and/or antibody-derived proteins, polypeptides, or
fragments or the like, including all of those described above.
Among very particularly preferred fusion proteins of the invention
in this regard are fusion proteins comprising an antibody or
antibody-derived protein or fragment such as those described above
and a ligand-binding moiety, such as those illustratively described
herein.
[0228] b. Target Binding Proteins
[0229] Also among preferred proteins of the invention in this
regard are antibodies and other types of target binding proteins,
and proteins relating thereto or derived therefrom, and protein
ligands, and proteins derived therefrom or relating thereto. Among
especially preferred ligand-binding proteins in this regard are
proteins that bind signal and effector proteins, and proteins
relating thereto or derived therefrom.
[0230] Among such binding proteins, including antibodies, including
proteins derived therefrom and proteins related thereto, are those
that bind to one or more of the following, alone or in any
combination: [0231] (i) CD proteins including but not limited to
CD3, CD4, CD8, CD19, CD20, and CD34; [0232] (ii) HER receptor
family proteins, including, for instance, HER2, HER3, HER4, and the
EGF receptor; [0233] (iii) cell adhesion molecules, for example,
LFA-1, Mol, p150,95, VLA-4, ICAM-1, VCAM, and alpha v/beta 3
integrin; [0234] (iv) growth factors, including but not limited to,
for example, vascular endothelial growth factor ("VEGF"); growth
hormone, thyroid stimulating hormone, follicle stimulating hormone,
luteinizing hormone, growth hormone releasing factor, parathyroid
hormone, mullerian-inhibiting substance, human macrophage
inflammatory protein (MIP-1 alpha), erythropoietin (EPO), nerve
growth factor, such as NGF-beta, platelet-derived growth factor
(PDGF), fibroblast growth factors, including, for instance, aFGF
and bFGF, epidermal growth factor (EGF), transforming growth
factors (TGF), including, among others, TGF-alpha and TGF-beta,
including TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, or TGF-beta5,
insulin-like growth factors-I and -II (IGF-I and IGF-II),
des(1-3)-IGF-I (brain IGF-I), and osteoinductive factors; [0235]
(v) insulins and insulin-related proteins, including but not
limited to insulin, insulin A-chain, insulin B-chain, proinsulin,
and insulin-like growth factor binding proteins; [0236] (vi)
coagulation and coagulation-related proteins, such as, among
others, factor VIII, tissue factor, von Willebrands factor, protein
C, alpha-1-antitrypsin, plasminogen activators, such as urokinase
and tissue plasminogen activator ("t-PA"), bombazine, thrombin, and
thrombopoietin; [0237] (vii) colony stimulating factors (CSFs),
including the following, among others, M-CSF, GM-CSF, and G-CSF;
[0238] (viii) other blood and serum proteins, including but not
limited to albumin, IgE, and blood group antigens; [0239] (ix)
receptors and receptor-associated proteins, including, for example,
flk2/flt3 receptor, obesity (OB) receptor, growth hormone
receptors, and T-cell receptors; [0240] (x) neurotrophic factors,
including but not limited to, bone-derived neurotrophic factor
(BDNF) and neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or
NT-6); [0241] (xi) relaxin A-chain, relaxin B-chain, and
prorelaxin; [0242] (xii) interferons, including for example,
interferon-alpha, -beta, and -gamma; [0243] (xiii) interleukins
(ILs), e.g., IL-1 to IL-10; [0244] (xiv) viral antigens, including
but not limited to, an AIDS envelope viral antigen; [0245] (xv)
lipoproteins, calcitonin, glucagon, atrial natriuretic factor, lung
surfactant, tumor necrosis factor-alpha and -beta, enkephalinase,
RANTES (regulated on activation normally T-cell expressed and
secreted), mouse gonadotropin-associated peptide, Dnase, inhibin,
and activin; [0246] (xvi) integrin, protein A or D, rheumatoid
factors, immunotoxins, bone morphogenetic protein (BMP), superoxide
dismutase, surface membrane proteins, decay accelerating factor
(DAF), AIDS envelope, transport proteins, homing receptors,
addressins, regulatory proteins, immunoadhesins, antibodies; and
[0247] (xvii) biologically active fragments or variants of any of
the foregoing.
[0248] As to all of the foregoing, particularly preferred are those
that are effective therapeutic agents, particularly those that
exert a therapeutic effect by binding a target, particularly a
target among those listed above, including targets derived
therefrom, targets related thereto, and modifications thereof.
[0249] c. Particular Illustrative Proteins
[0250] Among particular illustrative proteins are certain antibody
and antibody-related proteins, including peptibodies, such as, for
instance, those listed immediately below and elsewhere herein:
[0251] OPGL specific antibodies and peptibodies and the like (also
referred to as RANKL specific antibodies, peptibodies and the
like), including fully humanized and human OPGL specific
antibodies, particularly fully humanized monoclonal antibodies,
including but not limited to the antibodies described in
International Publication Number WO 03/002713, which is
incorporated herein in its entirety as to OPGL specific antibodies
and antibody related proteins, particularly those having the
sequences set forth therein, particularly, but not limited to,
those denoted therein: 9H7; 18B2; 2D8; 2E11; 16E1; and 22B3,
including the OPGL specific antibodies having either the light
chain of SEQ ID NO: 2 as set forth therein in FIG. 2 and/or the
heavy chain of SEQ ID NO:4, as set forth therein in FIG. 4, each of
which is individually and specifically incorporated by reference
herein in its entirety fully as disclosed in the foregoing
publication. Acid and base titrations of an OPGL specific antibody
("Ab-hOPGL") over the pH ranges of 4.5 to 5.0 and 5.0 to 5.5 are
described in the Examples below. The calculation of buffer capacity
of Ab-hOPGL in these pH ranges also is described in the Examples
below.
[0252] Myostatin binding agents or peptibodies, including myostatin
specific peptibodies, particularly those described in US
Application Publication Number 2004/0181033, which is incorporated
by reference herein in its entirely particularly in parts pertinent
to myostatin specific peptibodies, including but not limited to
peptibodies of the mTN8-19 family, including those of SEQ ID NOS:
305-351, including TN8-19-1 through TN8-19-40, TN8-19 con1 and
TN8-19 con2; peptibodies of the mL2 family of SEQ ID NOS: 357-383;
the mL15 family of SEQ ID NOS: 384-409; the mL17 family of SEQ ID
NOS: 410-438; the mL20 family of SEQ ID NOS: 439-446; the mL21
family of SEQ ID NOS: 447-452; the mL24 family of SEQ ID NOS:
453-454; and those of SEQ ID NOS: 615-631, each of which is
individually and specifically incorporated by reference herein in
its entirety fully as disclosed in the foregoing publication.
[0253] IL-4 receptor specific antibodies, particularly those that
inhibit activities mediated by binding of IL-4 and/or IL-13 to the
receptor, including those described in International Publication
No. WO 2005/047331 of International Application Number
PCT/US2004/03742, which is incorporated herein by reference in its
entirety particularly in parts pertinent to IL-4 receptor specific
antibodies, particularly such antibodies as are described therein,
particularly, and without limitation, those designated therein:
L1H1; L1H2; L1H3; L1H4; L1H5; L1H6; L1H7; L1H8; L1H9; L1H10; L1H11;
L2H1; L2H2; L2H3; L2H4; L2H5; L2H6; L2H7; L2H8; L2H9; L2H10; L2H11;
L2H12; L213; L2H14; L3H1; L4H1; L5H1; L6H1, each of which is
individually and specifically incorporated by reference herein in
its entirety fully as disclosed in the foregoing publication. Acid
and base titrations over the pH ranges of 4.5 to 5.0 and 5.0 to
5.5, and the calculation of buffer capacity in this range of an
IL-4 receptor specific antibody ("Ab-hIL4R") are described in the
Examples below.
[0254] Interleukin 1--receptor 1 ("IL1-R1") specific antibodies,
peptibodies and related proteins and the like, including but not
limited to those described in U.S. Application Publication Number
US2004/097712A1 which is incorporated herein by reference in its
entirety in parts pertinent to IL1-R1 specific binding proteins,
monoclonal antibodies in particular, especially, without
limitation, those designated therein: 15CA, 26F5, 27F2, 24E12, and
10H7, each of which is individually and specifically incorporated
by reference herein in its entirety fully as disclosed in the
aforementioned U.S. application publication.
[0255] Ang2 specific antibodies and peptibodies and related
proteins and the like, including but not limited to those described
in International Publication Number WO 03/057134 and U.S.
Application Publication Number US2003/0229023, each of which is
incorporated herein by reference in its entirety particularly in
parts pertinent to Ang2 specific antibodies and peptibodies and the
like, especially those of sequences described therein and including
but not limited to: L1(N); L1(N) WT; L1(N) 1K WT; 2.times.L1(N);
2.times.L1(N) WT; Con4 (N), Con4 (N) 1K WT, 2.times.Con4 (N) 1K;
L1(C); L1(C) 1K; 2.times.L1(C); Con4 (C); Con4 (C) 1K; 2.times.Con4
(C) 1K; Con4-L1(N); Con4-L1(C); TN-12-9 (N); C17 (N); TN8-8(N);
TN8-14 (N); Con 1 (N), also including anti-Ang 2 antibodies and
formulations such as those described in International Publication
Number WO 2003/030833 which is incorporated herein by reference in
its entirety as to the same, particularly Ab526; Ab528; Ab531;
Ab533; Ab535; Ab536; Ab537; Ab540; Ab543; Ab544; Ab545; Ab546;
A551; Ab553; Ab555; Ab558; Ab559; Ab565; AbF1AbFD; AbFE; AbFJ;
AbFK; AbG1D4; AbGC1E8; AbH1C12; Ab1A1; Ab1F; Ab1KAb1P; and Ab1P, in
their various permutations as described therein, each of which is
individually and specifically incorporated by reference herein in
its entirety fully as disclosed in the foregoing publication.
[0256] NGF specific antibodies, including, in particular, but not
limited to those described in US Application Publication Number
US2005/0074821, which is incorporated herein by reference in its
entirety particularly as to NGF-specific antibodies and related
proteins in this regard, including in particular, but not limited
to, the NGF-specific antibodies therein designated 4D4, 4G6, 6H9,
7H2, 14D10 and 14D11, each of which is individually and
specifically incorporated by reference herein in its entirety fully
as disclosed in the foregoing publication.
[0257] CD22 specific antibodies and related proteins, such as those
described in U.S. Pat. No. 5,789,554 which is incorporated herein
by reference in its entirety as to CD22 specific antibodies and
related proteins, particularly human CD22 specific antibodies, such
as but not limited to humanized and fully human antibodies,
including but not limited to humanized and fully human monoclonal
antibodies, particularly including but not limited to human CD22
specific IgG antibodies, such as, for instance, a dimer of a
human-mouse monoclonal hLL2 gamma-chain disulfide linked to a
human-mouse monoclonal hLL2 kappa-chain, including, but limited to,
for example, the human CD22 specific fully humanized antibody in
Epratuzumab, CAS registry number 501423-23-0. Illustrative of the
invention, acid and base titrations of a CD22-specific antibody
("Ab-hCD22") over the pH ranges of 4.5 to 5.0 and 5.0 to 5.5 are
described in the Examples below. The calculation of buffer capacity
of Ab-hCD22 in these pH ranges also is described in the Examples
below.
[0258] IGF-1 receptor specific antibodies and related proteins such
as those described in International Patent Application Number
PCT/US2005/046493, which is incorporated herein by reference in its
entirety as to IGF-1 receptor specific antibodies and related
proteins, including but not limited to the IGF-1 specific
antibodies therein designated L1H1, L2H2, L3H3, L4H4, L5H5, L6H6,
L7H7, L8H8, L9H9, L10H10, L11H11, L12H12, L13H13, L14H14, L15H15,
L16H16, L17H17, L18H18, L19H19, L20H20, L21H21, L22H22, L23H23,
L24H24, L25H25, L26H26, L27H27, L28H28, L29H29, L30H30, L31H31,
L32H32, L33H33, L34H34, L35H35, L36H36, L37H37, L38H38, L39H39,
L40H40, L41H41, L42H42, L43H43, L44H44, L45H45, L46H46, L47H47,
L48H48, L49H49, L50H50, L51H51, and L52H52, each of which is
individually and specifically incorporated by reference herein in
its entirety fully as disclosed in the foregoing International
Application.
[0259] B-7 related protein 1 ("B7RP-1") specific antibodies,
(B7RP-1 also is referred to in the literature as B7H2, ICOSL, B7h,
and CD275) particularly B7RP-specific fully human monoclonal IgG2
antibodies, particularly fully human IgG2 monoclonal antibody that
binds an epitope in the first immunoglobulin-like domain of B7RP-1,
especially those that inhibit the interaction of B7RP-1 with its
natural receptor, ICOS, on activated T cells in particular,
especially, in all of the foregoing regards, those disclosed in
U.S. Provisional Application No. 60/700,265, filed 18 Jul. 2005,
which is incorporated herein by reference in its entirety as to
such antibodies and related proteins, including but not limited to
antibodies designated therein as follow: 16H (having light chain
variable and heavy chain variable sequences SEQ ID NO:1 and SEQ ID
NO:7 respectively therein); 5D (having light chain variable and
heavy chain variable sequences SEQ ID NO:2 and SEQ ID NO:9
respectively therein); 2H (having light chain variable and heavy
chain variable sequences SEQ ID NO:3 and SEQ ID NO:10 respectively
therein); 43H (having light chain variable and heavy chain variable
sequences SEQ ID NO:6 and SEQ ID NO:14 respectively therein); 41H
(having light chain variable and heavy chain variable sequences SEQ
ID NO:5 and SEQ ID NO:13 respectively therein); and 15H (having
light chain variable and heavy chain variable sequences SEQ ID NO:4
and SEQ ID NO:12 respectively therein), each of which is
individually and specifically incorporated by reference herein in
its entirety fully as disclosed in the foregoing U.S. Provisional
Application. Acid and base titrations and determination of buffer
capacity of a B7RP-1 specific antibody ("Ab-hB7RP1") are
illustrated in the Examples below.
[0260] IL-15 specific antibodies, peptibodies and related proteins,
such as, in particular, humanized monoclonal antibodies,
particularly antibodies such as those disclosed in U.S. Application
Publication Numbers: US2003/0138421; US2003/023586; US2004/0071702,
each of which is incorporated herein by reference in its entirety
as to IL-15 specific antibodies and related proteins, including
peptibodies, including particularly, for instance, but not limited
to, HuMax IL-15 antibodies and related proteins, such as, for
instance, 146B7.
[0261] IFN gamma specific antibodies, especially human IFN gamma
specific antibodies, particularly fully human anti-IFN gamma
antibodies, such as, for instance, those described in US
Application Publication Number US2005/0004353, which is
incorporated herein by reference in its entirety as to IFN gamma
specific antibodies, particularly, for example, the antibodies
therein designated 1118; 1118*; 1119; 1121; and 1121* each of which
is individually and specifically incorporated by reference herein
in its entirety fully as disclosed in the foregoing US Application
Publication.
[0262] TALL-1 specific antibodies and other TALL specific binding
proteins such as those described in U.S. Application Publication
Number 2003/0195156 which is incorporated herein by reference in
its entirety as to TALL-1 binding proteins, particularly the
molecules of Tables 4 and SB, each of which is individually and
specifically incorporated by reference herein in its entirety fully
as disclosed in the foregoing US Application Publication.
[0263] Stem Cell Factor (s) ("SCF") and related proteins such as
those described in U.S. Pat. Nos. 6,204,363 and 6,207,802, each of
which is incorporated herein by reference in its entirety as to
stem cell factors and related proteins, particularly, for example,
the stem cells factor "STEMGEN.TM.."
[0264] Flt3-Ligands, ("Flt3L") and related proteins such as those
described in U.S. Pat. No. 6,632,424 which is incorporated herein
by reference as to Flt3-ligands and related proteins in this
regard.
[0265] IL-17 receptors and related proteins ("IL-17R"), such as
those described in U.S. Pat. No. 6,072,033 which is incorporated
herein by reference as to Flt3-ligands and related proteins in this
regard.
[0266] Etanercept, also referred to as Embre1, and related
proteins.
[0267] Actimmune (Interferon-gamma-1b), Activase (Alteplase),
Aldurazme (Laronidase), Amevive (Alefacept), Avonex (Interferon
beta-1a), BeneFIX (Nonacog alfa), Beromun (Tasonermin), Beatseron
(Interferon-beta-1b), BEXXAR (Tositumomab), Tev-Tropin
(Somatropin), Bioclate or RECOMBINATE (Recombinant), CEREZME
(Imiglucerase), ENBREL (Etanercept), Eprex (epoetin alpha),
EPOGEN/Procit (Epoetin alfa), FABRAZYME (Agalsidase beta),
Fasturtec/Elitek ELITEK (Rasburicase), FORTEO (Teriparatide),
GENOTROPIN (Somatropin), GlucaGen (Glucagon), Glucagon (Glucagon,
rDNA origin), GONAL-F (follitropin alfa), KOGENATE FS (Octocog
alfa), HERCEPTIN (Trastuzumab), HUMATROPE (SOMATROPIN), HUMIRA
(Adalimumab), Insulin in Solution, INFERGEN.RTM. (Interferon
alfacon-1), KINERET.RTM. (anakinra), Kogenate FS (Antihemophilic
Factor), LEUKIN (SARGRAMOSTIM Recombinant human
granulocyte-macrophage colony stimulating factor (rhuGM-CSF)),
CAMPATH (Alemtuzumab), RITUXAN.RTM. (Rituximab), TNKase
(Tenecteplase), MYLOTARG (gemtuzumab ozogamicin), NATRECOR
(nesiritide), ARANESP (darbepoetin alfa), NEULASTA (pegfilgrastim),
NEUMEGA (oprelvekin), NEUPOGEN (Filgrastim), NORDITROPIN CARTRIDGES
(Somatropin), NOVOSEVEN (Eptacog alfa), NUTROPIN AQ (somatropin),
Oncaspar (pegaspargase), ONTAK (denileukin diftitox), ORTHOCLONE
OKT (muromonab-CD3), OVIDREL (choriogonadotropin alfa), PEGASYS
(peginterferon alfa-2a), PROLEUKIN (Aldesleukin), PULMOZYME (domase
alfa), Retavase (Reteplase), REBETRON Combination Therapy
containing REBETOL.RTM. (Ribavirin) and INTRON.RTM. A (Interferon
alfa-2b), REBIF (interferon beta-1a), REFACTO (Antihemophilic
Factor), REFLUDAN (lepirudin), REMICADE (infliximab), REOPRO
(abciximab)ROFERON.RTM.-A (Interferon alfa-2a), SIMULECT
(baasiliximab), SOMAVERT (Pegivisomant), SYNAGIS.RTM.
(palivizumab), Stemben (Ancestim, Stem cell factor), THYROGEN,
INTRON.RTM. A (Interferon alfa-2b), PEG-INTRON.RTM. (Peginterferon
alfa-2b), XIGRIS.RTM. (Drotrecogin alfa activated), XOLAIR.RTM.
(Omalizumab), ZENAPAX.RTM. (daclizumab), ZEVALIN.RTM. (Ibritumomab
Tiuxetan).
[0268] d. Sequence Variation
[0269] Particularly preferred proteins in regard to all of the
foregoing and the following, include those that comprise a region
that is 70% or more, especially 80% or more, more especially 90% or
more, yet more especially 95% or more, particularly 97% or more,
more particularly 98% or more, yet more particularly 99% or more
identical in amino acid sequence to a reference amino acid sequence
of a binding protein, as illustrated above, particularly a
pharmaceutical binding protein, such as a GenBank or other
reference sequence of a reference protein.
[0270] Identity in this regard can be determined using a variety of
well-known and readily available amino acid sequence analysis
software. Preferred software includes those that implement the
Smith-Waterman algorithms, considered a satisfactory solution to
the problem of searching and aligning sequences. Other algorithms
also may be employed, particularly where speed is an important
consideration. Commonly employed programs for alignment and
homology matching of DNAs, RNAs, and polypeptides that can be used
in this regard include FASTA, TFASTA, BLASTN, BLASTP, BLASTX,
TBLASTN, PROSRCH, BLAZE, and MPSRCH, the latter being an
implementation of the Smith-Waterman algorithm for execution on
massively parallel processors made by MasPar.
[0271] The BLASTN, BLASTX, and BLASTP programs are among preferred
programs for such determinations, the former for polynucleotide
sequence comparisons and the latter two for polypeptide sequence
comparisons: BLASTX for comparison of the polypeptide sequences
from all three reading frames of polynucleotide sequence and BLASTP
for a single polypeptide sequence.
[0272] BLAST provides a variety of user definable parameters that
are set before implementing a comparison. Some of them are more
readily apparent than others on graphical user interfaces, such as
those provided by NCBI BLAST and other sequence alignment programs
that can be accessed on the internet. The settings and their values
are set out and explained on the service web sites and are
explained and set out in particular detail in a variety of readily
available texts, including but not limited to BIOINFORMATICS:
SEQUENCE AND GENOME ANALYSIS, 2.sup.nd Ed., David W. Mount, Cold
Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2004),
especially Chapters 3, 4, 5, and 6 as to comparison of protein and
nucleic acid sequences in general and as to BLAST comparisons and
searches in particular; SEQUENCE ANALYSIS IN A NUTSHELL: A GUIDE TO
COMMON TOOLS AND DATABASES, Scott Markel and Darryl Leon, O'Reilly
& Associates, Sebastopol, Calif. (2003), especially Chapter 7
as to BLAST in particular, each of which is herein incorporated by
reference in its entirety particularly in parts pertinent to
comparison of nucleotide and polypeptide sequences and to
determining their degree of identity, similarity, homology and/or
the like, especially as to comparison of a test sequence and a
reference sequence to calculate a degree (percent) of identity
between them.
[0273] In preferred embodiments of the invention in this regard,
relatedness of sequences is defined as the identity score in
percent returned by any one or another of the aforementioned BLAST
comparison searches with e=10 and all other parameters set to their
default values on the NCBI web server as set forth in SEQUENCE
ANALYSIS IN A NUTSHELL: A GUIDE TO COMMON TOOLS AND DATABASES,
Scott Markel and Darryl Leon, O'Reilly & Associates,
Sebastopol, Calif. (2003), pages 47-51 which are incorporated
herein by reference in their entireties and in all particulars of
the preferred settings for parameters of the present invention for
comparing sequences using BLAST, such as those on NCBI BLAST.
[0274] The following references provide additional information on
sequence comparisons in this regard, and in others. GUIDE TO HUMAN
GENOME COMPUTING, Ed. Martin J. Bishop, Academic Press, Harcourt
Brace & Company Publishers, New York (1994), which is
incorporated herein by reference in its entirety with regard to the
foregoing, particularly in parts pertinent to determining identity
and or homology of amino acid or polynucleotide sequences,
especially Chapter 7. The BLAST programs are described in Altschul
et al., "Basic Local Alignment Research Tool," J Mol Biol 215:
403-410 (1990), which is incorporated by reference herein in its
entirety. Additional information concerning sequence analysis and
homology and identity determinations are provided in, among many
other references well-known and readily available to those skilled
in the art: NUCLEIC ACID AND PROTEIN SEQUENCE ANALYSIS: A PRACTICAL
APPROACH, Eds. M. J. Bishop and C. J. Rawings, IRL Press, Oxford,
UK (1987); PROTEIN STRUCTURE: A PRACTICAL APPROACH, Ed. T. E.
Creighton, IRL Press, Oxford, UK (1989); Doolittle, R. F.:
"Searching through sequence databases," Met Enz. 183: 99-110
(1990); Meyers and Miller: "Optimal alignments in linear space"
Comput. Applica. in Biosci 4: 11-17 (1988); Needleman and Wunsch:
"A general method applicable to the search for similarities in
amino acid sequence of two proteins," J Mol Biol 48: 443-453 (1970)
and Smith and Waterman "Identification of common molecular
subsequences," J Mol Biol 147: 1950 et seq. (1981), each of which
is incorporated herein by reference in its entirety with reference
to the foregoing, particularly in parts pertinent to sequence
comparison and identity and homology determinations.
[0275] Particularly preferred embodiments in this regard have 50%
to 150% of the activity of the aforementioned reference protein,
particularly highly preferred embodiments in this regard have 60%
to 125% of the activity of the reference protein, yet more highly
preferred embodiments have 75% to 110% of the activity of the
reference protein, still more highly preferred embodiments have 85%
to 125% the activity of the reference, still more highly preferred
embodiments have 90% to 110% of the activity of the reference.
4. Formulations
[0276] Many reagents and methods conventionally employed for the
formulation of protein pharmaceuticals can be used for the
formulation of self-buffering protein compositions in accordance
with various aspects and preferred embodiments of the invention.
However, in self-buffering protein formulations in accordance with
the invention, buffering is provided substantially entirely by the
protein itself, not by a buffering agent, as is the case with
conventional formulations. Moreover, self-buffering protein
formulations in accordance with various aspects and preferred
embodiments of the invention are substantially free of such
buffering agents.
[0277] In many other respects, however, self-buffering protein
compositions in accordance with various aspects and embodiments of
the invention can be formulated using reagents and methods
conventionally employed for the formulation of proteins, in
particular, reagents and methods employed for the formulation of
pharmaceuticals, including pharmaceuticals for veterinary and human
use, especially those reagents and methods suitable for formulating
protein pharmaceuticals for veterinary and especially for human
use.
[0278] In accordance therewith, many methods and ingredients for
formulating and using pharmaceuticals that are well-known and
routine in the pertinent arts can be used in designing, making, and
using self-buffering protein formulations in accordance with
various aspects and preferred embodiments of the invention relating
thereto. Such methods and ingredients are described in, to name
just a few readily available references in this regard, REMINGTON:
THE SCIENCE AND PRACTICE OF PHARMACY, 21.sup.st Ed.; Beringer et
al. Editors, Lippincott, Williams & Wilkins, Philadelphia, Pa.
(2005); ANSEL'S PHARMACEUTICAL DOSAGE FORMS AND DRUG DELIVERY
SYSTEMS, 8.sup.th Ed., Allen et al., Editors, Lippincott, Williams
& Wilkins, Philadelphia, Pa. (2005); and PHARMACEUTICAL
FORMULATION OF PEPTIDES AND PROTEINS, Sven Frokjaer and Lars
Hovgaard, Editors, CRC Press, Boca Raton, Fla. (2000), each of
which is herein incorporated in its entirety particularly in parts
pertinent to conventional ingredients and methods that may be used
in self-buffering formulations of proteins in accordance with
various aspects and preferred embodiments of the invention relating
thereto.
[0279] Additional methods and ingredients that can be useful in
this regard are disclosed in, among others, U.S. Pat. No.
6,171,586; WO 2005/044854; U.S. Pat. No. 6,288,030; U.S. Pat. No.
6,267,958; WO 2004/055164; U.S. Pat. No. 4,597,966; US
2003/0138417; U.S. Pat. No. 6,252,055; U.S. Pat. No. 5,608,038;
U.S. Pat. No. 6,875,432; US 2004/0197324; WO 02/096457; U.S. Pat.
No. 5,945,098; U.S. Pat. No. 5,237,054; U.S. Pat. No. 6,485,932;
U.S. Pat. No. 6,821,515; U.S. Pat. No. 5,792,838; U.S. Pat. No.
5,654,403; U.S. Pat. No. 5,908,826; EP 0 804 163; and WO
2005/063291, each of which is incorporated herein by reference in
its entirety particularly in parts pertinent to pharmaceutically
acceptable self-buffering protein formulations in accordance with
the invention.
[0280] Various specific aspects of the ingredients and specific
types of formulations are further described below, by way of
illustration. The description thus provided is not exhaustive of
the methods and compositions possible for self-buffering protein
formulations in accordance with the various aspects and embodiments
of the invention, nor is it in any way exclusive.
[0281] In preferred embodiments of a variety of aspects of the
invention, formulations of self-buffering proteins comprise a
protein and a carrier, which also may be referred to herein
variously, as the case may be, as one or more of: a vehicle, a
primary vehicle, a diluent, a primary diluent, a primary carrier, a
solvent and/or a primary solvent. In the broadest sense the carrier
may be a gas, a liquid, or a solid, as suits the phase of the
composition and/or its use(s). In some embodiments of the invention
in this regard, the carrier is a solid, such as a powder in which a
protein may be dispersed. In preferred embodiments in this regard,
the carrier is a liquid, particularly a liquid in which the
self-buffering protein is highly soluble, particularly at
concentrations that provide the desired buffer capacity. Liquid
carriers may be organic or non-organic. Preferably they are
aqueous, most preferably they are largely or entirely comprised of
pure water.
[0282] It will be appreciated that formulations for pharmaceutical
use in accordance with various aspects and embodiments of the
invention must be compatible with the processes and conditions to
which they will be subjected, such as, for instance, sterilization
procedures (generally applied before mixing with an active agent),
and conditions during storage.
[0283] Almost invariably, formulations in accordance with numerous
aspects and embodiments of the invention will contain additional
ingredients including but not limited in any way to excipients and
other pharmaceutical agents. Nevertheless, it is to be understood
that formulations in accordance with the invention are
self-buffering formulations in which the buffer capacity is
provided substantially or entirely by the primary protein itself,
as described elsewhere herein.
[0284] Formulations in accordance with various aspects and
embodiments of the invention may contain, among others, excipients,
as described below, including but not limited to ingredients for
modifying, maintaining, or preserving, for example, osmolality,
osmolarity, viscosity, clarity, color, tonicity, odor, sterility,
stability, rate of dissolution or release, adsorption or
penetration of the formulations and/or primary polypeptide and/or
protein.
[0285] Formulations will, of course, depend upon, for example, the
particular protein being formulated, the other active agents, such
as other pharmaceuticals, that will be comprised in the
formulation, the intended route of administration, the method of
administration to be employed, the dosage, the dosing frequency,
and the delivery format, among others.
[0286] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
compositions comprising a protein preferably a pharmaceutical
protein and a solvent, the protein having a buffer capacity per
unit volume of at least that of approximately: 2.0 or 3.0 or 4.0 or
5.0 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or 40.0 or 50.0
or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or 350 or 400 or
500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or 3,000 or 4,000 or
5,000 mM sodium acetate buffer as determined over the range of pH
5.0 to 4.0 pH or 5.0 to 5.5 as described in Example 1 or 2 and
elsewhere herein.
[0287] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, wherein, exclusive of the buffer capacity of
the protein, the buffer capacity per unit volume of the composition
is equal to or less than that of 1.0 or 1.5 or 2.0 or 3.0 or 4.0 or
5.0 mM sodium acetate buffer as determined over the range of pH 5.0
to 4.0 or pH 5.0 to 5.5 as described in Example 1 or 2 and
elsewhere herein.
[0288] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, wherein
at the pH of the composition the buffer capacity of the protein is
at least approximately: 1.00 or 1.50 or 1.63 or 2.00 or 3.00 or
4.00 or 5.00 or 6.50 or 8.00 or 10.0 or 15.0 or 20.0 or 30.0 or
40.0 or 50.0 or 75.0 or 100 or 125 or 150 or 200 or 250 or 300 or
350 or 400 or 500 or 700 or 1,000 or 1,500 or 2,000 or 2,500 or
3,000 or 4,000 or 5,000 mEq per liter and per change in pH of one
pH unit.
[0289] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, wherein
at the pH of the composition, exclusive of the protein, the buffer
capacity per unit volume of the composition is equal to or less
than that of a 0.50 or 1.00 or 1.50 or 2.00 or 3.00 or 4.00 or 5.00
or 6.50 or 8.00 or 10.0 or 20.0 or 25.0 mM acetate buffer as
determined over the range of pH 5.0 to 4.0 or pH 5.0 to 5.5 as
described in Example 1 or 2 and elsewhere herein.
[0290] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, wherein
at a desired pH, the protein provides at least approximately 55%,
60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 99.5% of
the buffer capacity of the composition.
[0291] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, A,
wherein the concentration of the protein is between approximately:
20 and 400, or 20 and 300, or 20 and 250, or 20 and 200, or 20 and
150 mg/ml.
[0292] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, wherein
the pH maintained by the buffering action of the protein is a pH
between approximately: 3.5 and 8.0, or 4.0 and 6.0, or 4.0 and 5.5,
or 4.5 and 5.5.
[0293] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, wherein
the salt concentration is less than: 150 mM or 125 mM or 100 mM or
75 mM or 50 mM or 25 mM.
[0294] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, and
further comprising one or more pharmaceutically acceptable salts;
osmotic balancing agents (tonicity agents); anti-oxidants;
antibiotics; antimycotics; bulking agents; lyoprotectants;
anti-foaming agents; chelating agents; preservatives; colorants;
analgesics; or additional pharmaceutical agents.
[0295] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, and
further comprising one or more pharmaceutically acceptable polyols
in an amount that is hypotonic, isotonic, or hypertonic, preferably
approximately isotonic, particularly preferably isotonic,
especially preferably any one or more of sorbitol, mannitol,
sucrose, trehalose, or glycerol, particularly especially preferably
approximately 5% sorbitol, 5% mannitol, 9% sucrose, 9% trehalose,
or 2.5% glycerol, very especially in this regard 5% sorbitol, 5%
mannitol, 9% sucrose, 9% trehalose, or 2.5% glycerol.
[0296] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, and
further comprising one or more pharmaceutically acceptable
surfactants, preferably one or more of polysorbate 20, polysorbate
80, other fatty acid esters of sorbitan, polyethoxylates, and
poloxamer 188, particularly preferably polysorbate 20 or
polysorbate 80, preferably approximately 0.001 to 0.1% polysorbate
20 or polysorbate 80, very preferably approximately 0.002 to 0.02%
polysorbate 20 or polysorbate 80, especially 0.002 to 0.02%
polysorbate 20 or polysorbate 80.
[0297] Formulations in accordance with certain of the preferred
embodiments in various aspects of the invention provide
self-buffering protein compositions, particularly pharmaceutical
protein compositions, comprising a protein and a solvent, wherein
the protein is a pharmaceutical agent and the composition is a
sterile formulation thereof suitable for treatment of a veterinary
or a human medical subject.
[0298] Also among formulations in accordance with various aspects
and embodiments of the invention herein described are lyophilized
compositions in accordance with the foregoing, particularly
lyophilized compositions that when reconstituted provide a
formulation as described above and elsewhere herein.
[0299] a. Excipients and Other Additional Ingredients
[0300] As discussed above, certain embodiments in accordance with
aspects of the invention provide self-buffering protein
compositions, particularly pharmaceutical protein compositions,
that comprise, in addition to the protein, particularly a
pharmaceutical protein, one or more excipients such as those
illustratively described in this section and elsewhere herein.
Excipients can be used in the invention in this regard for a wide
variety of purposes, such as adjusting physical, chemical, or
biological properties of formulations, such as adjustment of
viscosity, and or processes of the invention to improve
effectiveness and or to stabilize such formulations and processes
against degradation and spoilage due to, for instance, stresses
that occur during manufacturing, shipping, storage, pre-use
preparation, administration, and thereafter.
[0301] A variety of expositions are available on protein
stabilization and formulation materials and methods useful in this
regard, such as Arakawa et al., "Solvent interactions in
pharmaceutical formulations," Pharm Res. 8(3): 285-91 (1991);
Kendrick et al., "Physical stabilization of proteins in aqueous
solution," in: RATIONAL DESIGN OF STABLE PROTEIN FORMULATIONS:
THEORY AND PRACTICE, Carpenter and Manning, eds. Pharmaceutical
Biotechnology. 13: 61-84 (2002), and Randolph et al.,
"Surfactant-protein interactions," Pharm Biotechnol. 13: 159-75
(2002), each of which is herein incorporated by reference in its
entirety, particularly in parts pertinent to excipients and
processes of the same for self-buffering protein formulations in
accordance with the current invention, especially as to protein
pharmaceutical products and processes for veterinary and/or human
medical uses.
[0302] Various excipients useful in the invention are listed in
Table 1 and further described below.
TABLE-US-00001 TABLE 1 Types of Excipients and Their Functions
Function Type Liquids Lyophilates Tonicity Provides isotonicity to
the formulation Stabilizers include cryo and Agents/ such that it
is suitable for injection lyoprotectants Stabilizers Examples
include polyols, salts, and Examples include polyols, sugars and
amino acids polymers Help maintain the protein in a more
Cryoprotectants protect proteins from compact state (polyols)
freezing stresses Minimize electrostatic, solution protein-
Lyoprotectants stabilize proteins in the protein interactions
(salts) freeze-dried state Bulking Not applicable Used to enhance
product elegance and to Agents prevent blowout Provides structural
strength to the lyo cake Examples include mannitol and glycine
Surfactants Prevent/control aggregation, particle Employed if
aggregation during the formation and surface adsorption of drug
lyophilization process is an issue Examples include polysorbate 20
and 80 May serve to reduce reconstitution times Examples include
polysorbate 20 and 80 Anti-oxidants Control protein oxidation
Usually not employed, molecular reactions in the lyophilized cake
are greatly retarded Metal A specific metal ion is included in a
May be included if a specific metal ion is Ions/ liquid formulation
only as a co-factor included only as a co-factor Chelating Divalent
cations such as zinc and Chelating agents are generally not Agents
magnesium are utilized in suspension needed in lyophilized
formulations formulations Chelating agents are used to inhibit
heavy metal ion catalyzed reactions Preservatives Important
particularly for multi-dose For multi-dose formulations only
formulations Provides protection against microbial Protects against
microbial growth, growth in formulation Example: benzyl alcohol Is
usually included in the reconstitution diluent (e.g. bWFI)
[0303] i. Salts
[0304] Salts may be used in accordance with certain of the
preferred embodiments of the invention to, for example, adjust the
ionic strength and/or the isotonicity of a self-buffering
formulation and/or to improve the solubility and/or physical
stability of a self-buffering protein or other ingredient of a
self-buffering protein composition in accordance with the
invention.
[0305] As is well known, ions can stabilize the native state of
proteins by binding to charged residues on the protein's surface
and by shielding charged and polar groups in the protein and
reducing the strength of their electrostatic interactions,
attractive, and repulsive interactions. Ions also can stabilize the
denatured state of a protein by binding to, in particular, the
denatured peptide linkages (--CONH) of the protein. Furthermore,
ionic interaction with charged and polar groups in a protein also
can reduce intermolecular electrostatic interactions and, thereby,
prevent or reduce protein aggregation and insolubility.
[0306] Ionic species differ significantly in their effects on
proteins. A number of categorical rankings of ions and their
effects on proteins have been developed that can be used in
formulating self-buffering protein compositions in accordance with
the invention. One example is the Hofmeister series, which ranks
ionic and polar non-ionic solutes by their effect on the
conformational stability of proteins in solution. Stabilizing
solutes are referred to as "kosmotropic." Destabilizing solutes are
referred to as chaotropic. Kosmotropes commonly are used at high
concentrations (e.g., >1 molar ammonium sulfate) to precipitate
proteins from solution ("salting-out"). Chaotropes commonly are
used to denture and/or to solubilize proteins ("salting-in"). The
relative effectiveness of ions to "salt-in" and "salt-out" defines
their position in the Hofmeister series.
[0307] In addition to their utilities and their drawbacks (as
discussed above) salts also are effective for reducing the
viscosity of protein formulations and can be used in the invention
for that purpose.
[0308] In order to maintain isotonicity in a parenteral formulation
in accordance with preferred embodiments of the invention, improve
protein solubility and/or stability, improve viscosity
characteristics, avoid deleterious salt effects on protein
stability and aggregation, and prevent salt-mediated protein
degradation, the salt concentration in self-buffering formulations
in accordance with various preferred embodiments of the invention
are less than 150 mM (as to monovalent ions) and 150 mEq/liter for
multivalent ions. In this regard, in certain particularly preferred
embodiments of the invention, the total salt concentration is from
about 75 mEq/L to about 140 mEq/L.
[0309] ii. Amino Acids
[0310] Free amino acids can be used in protein formulations in
accordance with various preferred embodiments of the invention as,
to name a few, bulking agents, stabilizers and antioxidants.
However, amino acids comprised in self-buffering protein
formulations in accordance with the invention do not provide
buffering action. For this reason, those with significant buffer
capacity either are not employed, are not employed at any pH around
which they have significant buffering activity, or are used at low
concentration so that, as a result, their buffer capacity in the
formulation is not significant. This is particularly the case for
histidine and other amino acids that commonly are used as buffers
in pharmaceutical formulations.
[0311] Subject to the foregoing consideration, lysine, proline,
serine, and alanine can be used for stabilizing proteins in a
formulation. Glycine is useful in lyophilization to ensure correct
cake structure and properties. As a result it is a common
ingredient in lyophilized formulations and reconstituted
lyophilates, such as Neumega.RTM., Genotropin.RTM., and
Humatrope.RTM.. Arginine may be useful to inhibit protein
aggregation, in both liquid and lyophilized formulations, such as
Activase.RTM., Avonex.RTM., and Enbrel.RTM. liquid. Methionine is
useful as an antioxidant.
[0312] iii. Polyols
[0313] Polyols include sugars, e.g., mannitol, sucrose, and
sorbitol and polyhydric alcohols such as, for instance, glycerol
and propylene glycol, and, for purposes of discussion herein,
polyethylene glycol (PEG) and related substances. Polyols are
kosmotropic. They are useful stabilizing agents in both liquid and
lyophilized formulations to protect proteins from physical and
chemical degradation processes. Polyols also are useful for
adjusting the tonicity of formulations.
[0314] Among polyols useful in the invention in this regard, is
mannitol, commonly used to ensure structural stability of the cake
in lyophilized formulations, such as, for example Leukine.RTM.,
Enbrel.RTM.--Lyo, and Betaseron.RTM.. It ensures structural
stability to the cake. It is generally used with a lyoprotectant,
e.g., sucrose. Sorbitol and sucrose are among preferred agents for
adjusting tonicity and as stabilizers to protect against
freeze-thaw stresses during transport or the preparation of bulks
during the manufacturing process. Reducing sugars (which contain
free aldehyde or ketone groups), such as glucose and lactose, can
glycate surface lysine and arginine residues. Therefore, they
generally are not among preferred polyols for use in accordance
with the invention. In addition, sugars that form such reactive
species, such as sucrose, which is hydrolyzed to fructose and
glucose under acidic conditions, and consequently engenders
glycation, also is not among preferred amino acids of the invention
in this regard. PEG is useful to stabilize proteins and as a
cryoprotectant and can be used in the invention in this regard,
such as it is in Recombinate.RTM..
[0315] iv. Surfactants
[0316] Protein molecules are susceptible to adsorption on surfaces
and to denaturation and consequent aggregation at air-liquid,
solid-liquid, and liquid-liquid interfaces. These effects generally
scale inversely with protein concentration. These deleterious
interactions generally scale inversely with protein concentration
and typically are exacerbated by physical agitation, such as that
generated during the shipping and handling of a product.
[0317] Surfactants routinely are used to prevent, minimize, or
reduce surface adsorption. Useful surfactants in the invention in
this regard include polysorbate 20, polysorbate 80, other fatty
acid esters of sorbitan polyethoxylates, and poloxamer 188.
[0318] Surfactants also are commonly used to control protein
conformational stability. The use of surfactants in this regard is
protein-specific since, any given surfactant typically will
stabilize some proteins and destabilize others.
[0319] Polysorbates are susceptible to oxidative degradation and
often, as supplied, contain sufficient quantities of peroxides to
cause oxidation of protein residue side-chains, especially
methionine. Consequently, polysorbates should be used carefully,
and when used, should be employed at their lowest effective
concentration. In this regard, polysorbates exemplify the general
rule that excipients should be used in their lowest effective
concentrations.
[0320] v. Antioxidants
[0321] A variety of processes can result in harmful oxidation of
proteins in pharmaceutical formulations. To some extent deleterious
oxidation of proteins can be prevented in pharmaceutical
formulations by maintaining proper levels of ambient oxygen and
temperature and by avoiding exposure to light. Antioxidant
excipients can be used as well to prevent oxidative degradation of
proteins. Among useful antioxidants in this regard are reducing
agents, oxygen/free-radical scavengers, and chelating agents.
Antioxidants for use in therapeutic protein formulations in
accordance with the invention preferably are water-soluble and
maintain their activity throughout the shelf life of a product.
EDTA is a preferred antioxidant in accordance with the invention in
this regard and can be used in the invention in much the same way
it has been used in formulations of acidic fibroblast growth factor
and in products such as Kineret.RTM. and Ontak.RTM..
[0322] Antioxidants can damage proteins. For instance, reducing
agents, such as glutathione in particular, can disrupt
intramolecular disulfide linkages. Thus, antioxidants for use in
the invention are selected to, among other things, eliminate or
sufficiently reduce the possibility of themselves damaging proteins
in the formulation.
[0323] vi. Metal Ions
[0324] Formulations in accordance with the invention may include
metal ions that are protein co-factors and that are necessary to
form protein coordination complexes, such as zinc necessary to form
certain insulin suspensions. Metal ions also can inhibit some
processes that degrade proteins. However, metal ions also catalyze
physical and chemical processes that degrade proteins.
[0325] Magnesium ions (10-120 mM) can be used to inhibit
isomerization of aspartic acid to isoaspartic acid. Ca.sup.+2 ions
(up to 100 mM) can increase the stability of human
deoxyribonuclease (rhDNase, Pulmozyme.RTM.). Mg.sup.+2, Mn.sup.+2,
and Zn.sup.+2, however, can destabilize rhDNase. Similarly,
Ca.sup.+2 and Sr.sup.+2 can stabilize Factor VIII, it can be
destabilized by Mg.sup.+2, Mn.sup.+2 and Zn.sup.+2, Cu.sup.+2 and
Fe.sup.+2, and its aggregation can be increased by Al.sup.+3
ions.
[0326] vii. Preservatives
[0327] Preservatives are necessary when developing multi-dose
parenteral formulations that involve more than one extraction from
the same container. Their primary function is to inhibit microbial
growth and ensure product sterility throughout the shelf-life or
term of use of the drug product. Commonly used preservatives
include benzyl alcohol, phenol and m-cresol. Although preservatives
have a long history of use with small-molecule parenterals, the
development of protein formulations that includes preservatives can
be challenging. Preservatives almost always have a destabilizing
effect (aggregation) on proteins, and this has become a major
factor in limiting their use in multi-dose protein formulations. To
date, most protein drugs have been formulated for single-use only.
However, when multi-dose formulations are possible, they have the
added advantage of enabling patient convenience, and increased
marketability. A good example is that of human growth hormone (hGH)
where the development of preserved formulations has led to
commercialization of more convenient, multi-use injection pen
presentations. At least four such pen devices containing preserved
formulations of hGH are currently available on the market.
Norditropin.RTM. (liquid, Novo Nordisk), Nutropin AQ.RTM. (liquid,
Genentech) & Genotropin (lyophilized--dual chamber cartridge,
Pharmacia & Upjohn) contain phenol while Somatrope.RTM. (Eli
Lilly) is formulated with m-cresol.
[0328] Several aspects need to be considered during the formulation
and development of preserved dosage forms. The effective
preservative concentration in the drug product must be optimized.
This requires testing a given preservative in the dosage form with
concentration ranges that confer anti-microbial effectiveness
without compromising protein stability. For example, three
preservatives were successfully screened in the development of a
liquid formulation for interleukin-1 receptor (Type I) using
differential scanning calorimetry (DSC). The preservatives were
rank ordered based on their impact on stability at concentrations
commonly used in marketed products.
[0329] As might be expected, development of liquid formulations
containing preservatives are more challenging than lyophilized
formulations. Freeze-dried products can be lyophilized without the
preservative and reconstituted with a preservative containing
diluent at the time of use. This shortens the time for which a
preservative is in contact with the protein, significantly
minimizing the associated stability risks. With liquid
formulations, preservative effectiveness and stability have to be
maintained over the entire product shelf-life (.about.18 to 24
months). An important point to note is that preservative
effectiveness has to be demonstrated in the final formulation
containing the active drug and all excipient components.
[0330] Self-buffering protein formulations in accordance with the
invention, particularly self-buffering biopharmaceutical protein
formulations, generally will be designed for specific routes and
methods of administration, for specific administration dosages and
frequencies of administration, for specific treatments of specific
diseases, with ranges of bio-availability and persistence, among
other things,
[0331] Formulations thus may be designed in accordance with the
invention for delivery by any suitable route, including but not
limited to orally, aurally, opthalmically, rectally, and vaginally,
and by parenteral routes, including intravenous and intraarterial
injection, intramuscular injection, and subcutaneous injection.
[0332] b. Formulations for Parenteral Administration
[0333] Formulations for parenteral administration may be in the
form of aqueous or non-aqueous isotonic sterile injection solutions
or suspensions. These solutions and suspensions may be prepared
from sterile powders or granules using one or more of the carriers
or diluents mentioned for use in the formulations for oral
administration or by using other suitable dispersing or wetting
agents and suspending agents.
[0334] When parenteral administration is contemplated, the
therapeutic compositions for use in this invention may be in the
form of a pyrogen-free, parenterally acceptable aqueous solution
comprising the desired protein in a pharmaceutically acceptable
vehicle. A particularly suitable vehicle for parenteral injection
is sterile pure water in which the protein is formulated as a
sterile, isotonic self-buffering solution.
[0335] Such preparations may also involve the formulation of the
desired protein in the form of, among other things, injectable
microspheres, bio-erodible particles, polymeric compounds
(polylactic acid, polyglycolic acid), beads, or liposomes,
including those that provide for controlled or sustained release.
Such formulations may be introduced by implantable drug delivery
devices, among others.
[0336] Formulations for parenteral administration also may contain
substances that adjust the viscosity. such as carboxymethyl
cellulose, sorbitol, and dextran. Formulations may also contain
ingredients that increase solubility of the desired protein or
other ingredients and those that stabilize one or more such
ingredients, including in some cases, the self-buffering
protein.
[0337] c. Formulations for Pulmonary Administration
[0338] A pharmaceutical composition in accordance with certain
embodiments of the invention may be suitable for inhalation. For
pulmonary administration, the pharmaceutical composition may be
administered in the form of an aerosol or with an inhaler including
dry powder aerosol. For example, a binding agent may be formulated
as a dry powder for inhalation. Inhalation solutions may also be
formulated with a propellant for aerosol delivery. In yet another
embodiment, solutions may be nebulized. Pulmonary administration is
further described in PCT Application No. PCT/US94/001875, which
describes pulmonary delivery of chemically modified proteins.
[0339] d. Formulations for Oral Administration
[0340] For oral administration, the pharmaceutical composition may
be in the form of, for example, a tablet, capsule, suspension, or
liquid. The pharmaceutical composition is preferably made in the
form of a dosage unit containing a particular amount of the active
ingredient. Examples of such dosage units are tablets or capsules.
Formulations for oral administration in accordance with the
invention in this regard can be made conventionally wherein
buffering in the formulation is provided by the self-buffering
protein as described elsewhere herein.
[0341] e. Controlled Release Formulations
[0342] Among additional formulations that can be useful in the
invention as herein described are sustained- and
controlled-delivery formulations. Techniques for making such
sustained- and controlled-delivery formulations that may be used in
accordance with various aspects and preferred embodiments of the
invention are well-known to those skilled in the art. Among these
are delivery methods that use liposome carriers, bio-erodible
microparticles, porous beads, and semi-permeable polymer matrices,
such as those described in PCT/US93/00829; U.S. Pat. No. 3,773,919;
EP 58,481; Sidman et al., Biopolymers, 22:547-556 (1983); Langer et
al., J. Biomed. Mater. Res., 15:167-277, (1981); Langer et al.,
Chem. Tech., 12:98-105 (1982); EP 133,988; Eppstein et al., Proc.
Natl. Acad. Sci. (USA), 82:3688-3692 (1985); EP 36,676; EP 88,046;
and EP 143,949, each of which is hereby incorporated by reference
in its entirety, particularly in parts pertinent to self-buffering
sustained- and controlled-delivery pharmaceutical protein
formulations in accordance with the invention herein described.
[0343] f. Sterilization
[0344] The pharmaceutical composition to be used for in vivo
administration typically must be sterile. This may be accomplished
by filtration through sterile filtration membranes. Where the
composition is lyophilized, sterilization using this method may be
conducted either prior to or following lyophilization and
reconstitution. The composition for parenteral administration may
be stored in lyophilized form or in solution. In addition,
parenteral compositions generally are placed into a container
having a sterile access port, for example, an intravenous solution
bag or vial having a stopper pierceable by a hypodermic injection
needle.
[0345] g. Storage
[0346] Once the pharmaceutical composition has been formulated, it
may be stored in sterile vials as a solution, suspension, gel,
emulsion, solid, or a dehydrated or lyophilized powder. Such
formulations may be stored either in a ready-to-use form or in a
form (e.g., lyophilized) requiring reconstitution prior to
administration.
[0347] h. Additional Pharmaceutical Agents
[0348] Self-buffering protein compositions in accordance with the
invention, particularly self-buffering pharmaceutical protein
compositions, can comprise in addition to the self-buffering
protein of the composition, one or more additional pharmaceutical
agents. Such agents may be proteins as well, or they may be other
types of agents. Included among such agents are those for
prevention or treatment of any disorder or disease. Such agents
include, for instance, antibiotics and antimycotics. They also
include agents for treating human disorders, including but not
limited to, agents for treating inflammatory diseases, cancers,
metabolic disorders, neurological and renal disorders, to name just
a few. Agents that may be used in the invention in this regard also
include agents useful to augment the action of a self-buffering
composition and or prevent, ameliorate, or treat any undesirable
side effects of the administration thereof.
[0349] i. Methods for Making Self-Buffering Protein
Formulations
[0350] Compositions in accordance with the invention may be
produced using well-known, routine methods for making, formulating,
and using proteins, particularly pharmaceutical proteins. In
certain of the preferred embodiments of a number of aspects of the
invention in this regard, methods for preparing the compositions
comprise the use of counter ions to remove residual buffering
agents. In this regard the term counter ion is any polar or charged
constituent that acts to displace buffer from the composition
during its preparation. Counter ions useful in this regard include,
for instance, glycine, chloride, sulfate, and phosphate. The term
counter ion in this regard is used to mean much the same thing as
displacement ion.
[0351] Residual buffering agents can be removed using the counter
ions in this regard, using a variety of well-known methods,
including but not limited to, standard methods of dialysis and high
performance membrane diffusion-based methods such as tangential
flow diafiltration. Methods for residual buffer removal employing a
counter ion in this regard can also, in some cases, be carried out
using size exclusion chromatography.
[0352] In certain related preferred embodiments in this regard,
compositions in accordance with the invention are prepared by a
process that involves dialysis against a bufferless solution at a
pH below that of the preparation containing the self-buffering
protein. In particularly preferred embodiments of the invention in
this regard, the bufferless solution comprises counter ions,
particularly those that facilitate removal of residual buffer and
do not adversely affect the self-buffering protein or the
formulation thereof. In further particularly preferred embodiments
of the invention in this regard, following dialysis the pH of the
preparation is adjusted to the desired pH using dilute acid or
dilute base.
[0353] In certain related particularly preferred embodiments in
this regard, compositions in accordance with the invention are
prepared by a process that involves tangential flow diafiltration
against a bufferless solution at a pH below that of the preparation
containing the self-buffering protein. In particularly preferred
embodiments of the invention in this regard, the bufferless
solution comprises counter ions, particularly those that facilitate
removal of residual buffer and do not adversely affect the
self-buffering protein or the formulation thereof. In further
particularly preferred embodiments of the invention in this regard,
following diafiltration the pH of the preparation is adjusted to
the desired pH using dilute acid or dilute base.
5. Routes of Administration
[0354] Formulations in accordance with the invention, in various
embodiments, may be administered by a variety of suitable routes,
well-known to those skilled in the art of administering
therapeutics to a subject. In embodiments of the invention in this
regard, one or more formulations, as described elsewhere herein,
are administered via the alimentary canal. In other embodiments one
or more formulations as described elsewhere herein are administered
parenterally. In various embodiments one or more formulations may
be administered via the alimentary canal in conjunction with one or
more other formulations administered parenterally.
[0355] Such routes in a variety of embodiments include but are not
limited to administration of the compositions orally, ocularly,
mucosally, topically, rectally, pulmonarily, such as by inhalation
spray, and epicutaneously. The following parenteral routes of
administration also are useful in various embodiments of the
invention: administration by intravenous, intraarterial,
intracardiac, intraspinal, intrathecal, intraosseous,
intraarticular, intrasynovial, intracutaneous, intradermal,
subcutaneous, peritoneal, and/or intramuscular injection. In some
embodiments intravenous, intraarterial, intracutaneous,
intradermal, subcutaneous and/or intramuscular injection are used.
In some embodiments intravenous, intraarterial, intracutaneous,
subcutaneous, and/or intramuscular injection are used.
[0356] In certain embodiments of the invention the compositions are
administered locally, for instance by intraocular injection to
treat ocular neovascularization, retinopathy, or age-related
macular degeneration.
6. Doses
[0357] The amount of a self-buffering protein formulation
administered and the dosage regimen for treating a disease
condition with the formulation depends on a variety of factors,
including the age, weight, sex, and medical condition of the
subject, the type of disease, the severity of the disease, the
route and frequency of administration, and the particular
formulation employed. In particular the amount will depend on the
protein therapeutic being administered and any other therapeutic
agents being administered in conjunction therewith. Dosages can be
determined for formulations in accordance with the invention using
well-established routine pharmaceutical procedures for this
purpose.
7. Dosing Regimens
[0358] Formulations of the invention can be administered in dosages
and by techniques well-known to those skilled in the medical and
veterinary arts taking into consideration such factors as the age,
sex, weight, and condition of the particular patient, and the
formulation that will be administered (e.g., solid vs. liquid).
Doses for humans or other mammals can be determined without undue
experimentation by the skilled artisan, from this disclosure, the
documents cited herein, and the knowledge in the art.
[0359] In accordance with various embodiments, proper dosages and
dosing plans will depend on numerous factors, and may vary in
different circumstances. The parameters that will determine the
optimal dosage plans to be administered typically will include some
or all of the following: the disease being treated and its stage;
the species of the subject, their health, gender, age, weight, and
metabolic rate; other therapies being administered; and expected
potential complications from the subject's history or genotype.
[0360] The optimal dosing plan in a given situation also will take
into consideration the nature of the formulation, the way it is
administered, the distribution route following administration, and
the rate at which it will be cleared both from sites of action and
from the subject's body. Finally, the determination of optimal
dosing preferably will provide an effective dose that is neither
below the threshold of maximal beneficial effect nor above the
threshold where the deleterious effects associated with the dose of
the active agents outweighs the advantages of the increased
dose.
[0361] It will be appreciated that a "dose" may be delivered all at
once, fractionally, or continuously over a period of time. The
entire dose also may be delivered to a single location or spread
fractionally over several locations. Furthermore, doses may remain
the same over a treatment, or they may vary.
[0362] In various embodiments, formulations in accordance with the
invention are administered in an initial dose, and thereafter
maintained by further administrations. A formulation of the
invention in some embodiments is administered by one method
initially, and thereafter administered by the same method or by one
or more different methods. The dosages of on-going administrations
may be adjusted to maintain at certain values the levels of the
active agents in the subject. In some embodiments the compositions
are administered initially, and/or to maintain their level in the
subject, by intravenous injection. In a variety of embodiments,
other forms of administration are used.
[0363] Formulations of the invention may be administered in many
frequencies over a wide range of times, including any suitable
frequency and range of times that delivers a treatment-effective
dose. Doses may be continuously delivered, administered every few
hours, one or more times a day, every day, every other day or
several times a week, or less frequently. In some embodiments they
are administered over periods of one, two, three, four, five, six,
seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, or
more days. In some embodiments they are administered over periods
of one, two, three, four, five, six, seven, eight, nine, ten,
eleven, twelve, or more months. In a variety of embodiments they
are administered for a period of one, two, three, four, five, six,
seven, eight, nine, ten, or more years. Suitable regimens for
initial administration and further doses for sequential
administrations may all be the same or may be variable. Appropriate
regimens can be ascertained by the skilled artisan, from this
disclosure, the documents cited herein, and the knowledge in the
art. Generally lengths of treatment will be proportional to the
length of the disease process, the effectiveness of the therapies
being applied, and the condition and response of the subject being
treated.
8. Diseases and Treatments
[0364] Self-buffering pharmaceutical protein compositions in
accordance with the invention, in preferred embodiments, are useful
to treat subjects suffering from a wide variety of disorders and
diseases. As noted elsewhere herein, the invention provides, among
others, self-buffering compositions of pharmaceutical antibodies,
antibody-derived pharmaceutical proteins, and antibody-related
pharmaceutical proteins, that can comprise Fc effector functions
and binding domains specific for a wide variety of disease-related
targets and that are useful for treating disease. These proteins
and self-buffering compositions thereof are described at length
herein above, as well as their use in treating various disorders
and diseases associated with their targets. Methods for using the
compositions, including formulation methods, administration
methods, doses, and dosing methods are all described illustratively
above. The formulation and administration of any particular
composition of the invention can be tailored to the treatment of a
particular disease, using well-known and routine techniques in the
arts for doing so, taken in light of the guidance provided by the
present description of the invention. Among diseases usefully
treated using self-buffering pharmaceutical protein formulations in
accordance with various aspects and preferred embodiments of the
invention are inflammatory diseases, cancers, metabolic disorders,
neurological and renal disorders, to name just a few.
9. Packaging and Kits
[0365] The invention also provides kits comprising self-buffering
protein formulations, particularly kits comprising in one more
containers, a self-buffering pharmaceutical protein formulation and
instructions regarding the use thereof, particularly such kits
wherein the formulation is a pharmaceutically acceptable
formulation for human use. Among preferred kits are those
comprising one or more containers of a self-buffering protein
formulation of the invention and one or more separate documents,
information pertaining to the contents of the kit, and/or the use
of its contents, particularly those wherein the protein is a
biopharmaceutical protein, especially those wherein the protein is
a biopharmaceutical protein formulated for the treatment of a
disease in humans.
[0366] In certain aspects of the invention in this regard,
preferred kits include kits as above further comprising one or more
single or multi-chambered syringes (e.g., liquid syringes and
lyosyringes) for administering one or more self-buffering protein
formulations of the invention. In certain aspects of the invention
in this regard, certain of the particularly preferred kits further
comprise preloaded syringes. In further particularly preferred
embodiments in this regard, the kits comprise a self-buffering
pharmaceutical composition for parenteral administration, sealed in
a vial under partial vacuum in a form ready for loading into a
syringe and administration to a subject. In especially preferred
embodiments in this regard, the composition is disposed therein
under partial vacuum. In all of these regards and others, in
certain further particularly preferred embodiments the kits contain
one or more vials in accordance with any of the foregoing, wherein
each vial contains a single unit dose for administration to a
subject. In all these respects and others the invention further
relates to kits comprising lyophilates, disposed as above, that
upon reconstitution provide compositions in accordance therewith.
In this regard, the invention further provides in certain of its
preferred embodiments, kits that contain a lyophilate in accordance
with the invention and a sterile diluent for reconstituting the
lyophilate.
EXAMPLES
[0367] The present invention is additionally described by way of
the following illustrative, non-limiting Examples.
Example 1
Acid Titrations and Buffer Capacities of Sodium Acetate Buffers in
the Range pH 5.0 to 4.0
[0368] A stock solution of known concentration of acetic acid was
prepared by diluting ultrapure glacial acetic acid in HPLC grade
water and then titrating the pH up to the desired value with NaOH.
Stocks were equilibrated to the air and to 21.degree. C. Volumetric
standards were prepared at a concentration of 1 N and diluted as
necessary with HPLC water.
[0369] One mM, 2.5 mM, 5 mM, 7.5 mM, 10 mM, and 15 mM sodium
acetate buffers were prepared by diluting the stock in HPLC water.
The solutions were titrated with HCl. 0.2 N HCl was used for the 1,
2.5, and 5 mM solutions, 0.4 N HCl was used for the 7.5 mM
solution, and 0.8 N HCl was used for the 10 and 15 mM solutions.
The titrations were performed using standard analytical laboratory
techniques.
[0370] FIG. 1, Panel A shows the titration data and the least
squares trend lines calculated from the data for each solution. The
slope of the trend line calculated from each data set was taken as
the buffer capacity of the corresponding acetate buffer. The linear
dependence of buffer capacity on acetate buffer concentration is
shown in FIG. 1, Panel B.
Example 2
Base Titrations and Buffer Capacities of Sodium Acetate Buffers in
the Range pH 5.0 to 5.5
[0371] Acetate buffer stocks and solutions for titration were
prepared as described in Example 1. The solutions were titrated as
described in Example 1, except that the solutions were titrated
from pH 5.0 to 5.5 and the titrations were done using NaOH instead
of HCl. 0.2 N NaOH was used to titrate the 1, 2.5, and 5 mM
solutions and 0.4 N NaOH was used for the 7.5, 10, and 15 mM
solutions. The results of the titrations are shown in FIG. 2A. The
linear dependence of buffer capacity on concentration of acetate
buffer is displayed in FIG. 2B.
Example 3
Determination of Acetate by HPLC
[0372] Acetate was determined in acetate buffer samples using
analytical SE-HPLC. A standard curve for peak areas as a function
of acetate concentration was established by analysis of acetate in
buffers of known acetate concentration. The amount of acetate in
test samples was interpolated from the standard curve. A standard
curve is shown in FIG. 3. Nominal and measured amount of acetate in
test buffers are tabulated below the standard curve in the
figure.
Example 4
Acid Titrations of Ab-hOPGL Formulations Over the Range of pH 5.0
to pH 4.0
[0373] Bulk Ab-hOPGL in 10 mM acetate (nominal value), 5% sorbitol,
pH 5.0 was diafiltered against 5.25% sorbitol, pH 3.2 (adjusted
with HCl) in a LABSCALE TFF.RTM. system (Millipore) with a
multi-manifold cassette, using 3 Millipore Pellicon XL 50
regenerated cellulose ultra-filtration membranes. The diafiltration
solution was exchanged 8 to 10 times over the course of the
diafiltration for each formulation. Following diafiltration, the pH
of the resulting buffer-free solution was measured and adjusted to
pH 5.0, using 0.05 N HCl or 0.05 N NaOH.
[0374] One, 10, 30, 60, 90, and 110 mg/ml solutions were prepared
for titration by dilution. The pH of each dilution was adjusted to
pH 5.0 with NaOH or HCl as necessary. Titrations were carried out
as described in the foregoing Examples. 0.2 N HCl was used to
titrate the 1, 10, and 30 mg/ml solutions. 0.4 N HCl was used to
titrate the 60 mg/ml solution. 0.8 N HCl was used to titrate the 90
and 110 solutions.
[0375] The results of the titrations are depicted in FIG. 4. The
least squares regression line is shown for the dataset for each
concentration. The buffer capacity was taken as the slope of the
regression line for each concentration.
Example 5
Base Titrations of Ab-hOPGL Formulations Over the Range of pH 5.0
to 6.0
[0376] One, 10, 30, 60, 90, and 110 mg/ml solutions of Ab-hOPGL
were prepared for titration as described in Example 4. Base
titrations were carried out using NaOH as described in preceding
Examples. 0.2 N NaOH was used for the 1, 10, 30, and 60 mg/ml
solutions and 0.4 N NaOH was used for the 90 and 110 mg/ml
solutions. Results of the titrations are depicted in the graph in
FIG. 5. Linear regression lines are shown for the data for each
concentration. The buffer capacity was taken as the slope of the
regression line for each concentration.
Example 6
Residual Acetate Levels in Self-Buffering Ab-hOPGL Formulations
[0377] The amount of residual acetate was determined in Ab-hOPGL
formulations using the methods described in Example 3. The results
are depicted graphically in FIG. 6, which shows a standard curve
relating HPLC measurements to acetate concentrations and, below the
graph, a tabulation of the results of determinations made on
Ab-hOPGL formulations at different concentrations. Ab-hOPGL
concentrations are indicated on the left ("Nominal") and the
measured concentration of acetate in each of the Ab-hOPGL
concentration is indicated on the right.
Example 7
Buffer Capacity of Ab-hOPGL Formulations Plus or Minus Residual
Acetate in the Range of pH 5.0 to 4.0
[0378] Self-buffered Ab-hOPGL formulations were prepared and
titrated with HCl as described in foregoing Examples. In addition,
data was adjusted by subtracting the contribution of residual
acetate buffer based on the determination of acetate content by
SE-HPLC as described in, for instance, Example 3. Buffer capacities
were determined as described above. The same analysis was carried
out on both sets of data. The results, depicted in FIG. 7, show the
effect of residual acetate on the buffer capacity of the Ab-hOPGL
preparations. The results make it clear that the buffer capacity of
residual acetate is a minor factor in the buffer capacity of the
self-buffering Ab-hOPGL formulations that were analyzed.
Example 8
Buffer Capacity of Ab-hOPGL Plus or Minus Residual Acetate in the
Range of pH 5.0 to 6.0
[0379] Self-buffered Ab-hOPGL formulations were prepared and
titrated with NaOH as described in foregoing Examples. In addition,
data was adjusted by subtracting the contribution of residual
acetate buffer based on the determination of acetate content by
SE-HPLC as described in, for instance, Example 3. Buffer capacities
were determined as described above. The same analysis was carried
out on both sets of data. The results, depicted in FIG. 8, show the
effect of residual acetate on the buffer capacity of the Ab-hOPGL
preparations. The results make it clear that the buffer capacity of
residual acetate is a minor factor in the buffer capacity of the
self-buffering Ab-hOPGL formulations that were analyzed.
Example 9
pH and Ab-hOPGL Stability in Self-Buffered and Conventionally
Buffered Formulations
[0380] Self-buffering formulations of Ab-hOPGL were prepared as
described in the foregoing Examples. In addition, formulations were
made containing a conventional buffering agent, either acetate or
glutamate. All formulations contained 60 mg/ml Ab-hOPGL. The
stability of pH and Ab-hOPGL in the formulations was monitored for
six months of storage at 4.degree. C. Stability was monitored by
determining monomeric Ab-hOPGL in the formulations over the time
course of storage. The determination was made using SE-HPLC as
described above. The results for all three formulations are shown
in FIG. 9. Panel A shows the stability of Ab-hOPGL in the three
formulations. Stability in the self-buffered formulation is as good
as in the conventionally buffered formulations. Panel B shows the
pH stability of the three formulations. Again, pH stability in the
self-buffered formulation is as good as in the conventionally
buffered formulations.
Example 10
Titration and Buffer Capacities of Ab-hB7RP1--pH 5.0 to 4.0
[0381] Self-buffering formulations of Ab-hB7RP1 were prepared in
concentrations of 1, 10, 30, and 60 mg/ml, as described for
Ab-hOPGL in the foregoing Examples. Titrations were carried out
using HCl as described above. In addition, data was adjusted by
subtracting the contribution of residual acetate buffer based on
the determination of acetate content by SE-HPLC as described in,
for instance, Example 3. FIG. 10, Panel A shows the titration
results. FIG. 10, Panel B shows the dependence of buffer capacity
on the concentration of Ab-hB7RP1 formulations before and after
subtracting the contribution of residual acetate buffer. The
results clearly show the self-buffering capacity of Ab-hB7RP1 in
this pH range. At 40 mg/ml it provides approximately as much buffer
capacity in this pH range as 10 mM sodium acetate buffer. At 60
mg/ml it provides approximately as much buffer capacity as 15 mM
sodium acetate buffer.
Example 11
Titration and Buffer Capacities for Ab-hB7RP1--pH 5.0 to 6.0
[0382] Self-buffering formulations of Ab-hB7RP1 were prepared in
concentrations of 1, 10, 30, and 60 mg/ml, as described for
Ab-hOPGL in the foregoing Examples. Titrations were carried out
using NaOH as described above. In addition, data was adjusted by
subtracting the contribution of residual acetate buffer based on
the determination of acetate content by SE-HPLC as described in,
for instance, Example 3. FIG. 11, Panel A shows the titration
results. FIG. 11, Panel B shows the dependence of buffer capacity
on the concentration of Ab-hB7RP1 formulations before and after
subtracting the contribution of residual acetate buffer. The
results clearly show the self-buffering capacity of Ab-hB7RP1 in
this pH range. At 60 mg/ml it provides approximately as much buffer
capacity in this pH range as 10 mM sodium acetate buffer.
Example 12
Ab-hB7RP1 Stability in Self-Buffering and Conventionally Buffered
Formulations at 4.degree. C. and 29.degree. C.
[0383] Ab-hB7RP1 was prepared as described in the foregoing
Examples and formulated as described above, in self-buffering
formulations and in formulations using a conventional buffering
agent, either acetate or glutamate. All formulations contained 60
mg/ml Ab-hB7RP1. The stability of the solution's pH and of the
Ab-hB7RP1 in the solution was monitored for twenty-six weeks of
storage at 4.degree. C. or at 29.degree. C. Stability was monitored
by determining monomeric Ab-hB7RP1 in the formulations over the
time course of storage. The determination was made using SE-HPLC as
described above. The results are shown in FIG. 12. Panel A shows
the results for storage at 4.degree. C. Panel B shows the results
for storage at 29.degree. C. Ab-hB7RP1 was at least as stable in
the self-buffered formulation at 4.degree. C. as the conventionally
buffered formulations. At 29.degree. C. the self-buffered
formulation was at least as stable as the conventionally buffered
formulations, and may have been slightly better from 10 weeks
through the last time point.
Example 13
pH Stability of Self-Buffered Ab-hB7RP1 at 4.degree. C. and
29.degree. C.
[0384] Self-buffered Ab-hB7RP1 at 60 mg/ml was prepared as
described in the foregoing Example. pH was monitored over the time
course and at the same temperatures as described therein. The
results are shown in FIG. 13.
Example 14
Buffer Capacity of Ab-hCD22 Formulations--pH 4.0 to 6.0
[0385] Self-buffering formulations of Ab-hCD22 were prepared and
titrated over the range of pH 5.0 to 4.0 and the range of 5.0 to
6.0, as described for Ab-hOPGL and Ab-hB7RP1 in the foregoing
Examples. Buffer capacities were calculated from the titration
data, also as described above. Buffer capacity as a function of
concentration is shown in FIG. 14 for both pH ranges. Panel A shows
the buffer capacity of the Ab-hCD22 formulations over the range of
pH 5.0 to 4.0. Buffer capacity is linearally dependent on
concentration, and an approximately 21 mg/ml formulation of
Ab-hCD22 has a buffer capacity equal to that of 10 mM sodium
acetate buffer pH 5.0, measured in the same way. Panel B shows the
buffer capacity as a function of concentration over the pH range
5.0 to 6.0. In this range of pH an approximately 30 mg/ml
formulation of Ab-hCD22 has a buffer capacity equal to that of 10
mM sodium acetate buffer pH 5.0, measured in the same way.
Example 15
Titrations and Buffer Capacities of Ab-hIL4R Formulations--pH 5.0
to 4.0
[0386] Self-buffering formulations of Ab-hIL4R were prepared in
concentrations of 1, 10, 25, and 90 mg/ml, as described for
Ab-hOPGL in the foregoing Examples. Titrations were carried out
using HCl as described above. FIG. 15, Panel A shows the titration
results. FIG. 15, Panel B shows the dependence of buffer capacity
on the concentration of Ab-hIL4R. The results clearly show the
self-buffering capacity of Ab-hIL4R in this pH range. At
approximately 75 mg/ml it provides as much buffer capacity in this
pH range as 10 mM sodium acetate pH 5.0, measured in the same
way.
Example 16
Titrations and Buffer Capacities of Ab-hIL4R Formulations--pH 5.0
to 6.0
[0387] Self-buffering formulations of Ab-hIL4R were prepared in
concentrations of 1, 10, 25, and 90 mg/ml, as described for
Ab-hOPGL in the foregoing Examples. Titrations were carried out
using NaOH as described above. FIG. 16, Panel A shows the titration
results. FIG. 16, Panel B shows the dependence of buffer capacity
on the concentration of Ab-hIL4R in this pH range. The results
clearly show the self-buffering capacity of Ab-hIL4R in this pH
range. At approximately 90 mg/ml it provides as much buffer
capacity in this pH range as 10 mM sodium acetate pH 5.0, measured
in the same way.
Example 17
Ab-hIL4R and pH Stability in Acetate and Self-Buffered Ab-hIL4R
Formulations at 37.degree. C.
[0388] Self-buffered and acetate buffered formulations of Ab-hIL4R
at pH 5.0 and 70 mg/ml were prepared as described above. pH and
Ab-hIL4R stability were monitored in the formulations for 4 weeks
at 37.degree. C. Ab-hIL4R stability was monitored by SE-HPLC as
described above. The results are shown in FIG. 17. Panel A shows
that Ab-hIL4R is at least as stable in the self-buffered
formulation as in the sodium acetate buffer formulation. Panel B
shows that pH in the self-buffered formulation is as stable as in
the sodium acetate buffer formulation.
* * * * *