U.S. patent application number 14/187055 was filed with the patent office on 2016-01-07 for therapeutic protein compositions having reduced immunogenicity and/or improved efficacy.
This patent application is currently assigned to Barofold, Inc.. The applicant listed for this patent is Barofold, Inc., The Regents of the University of Colorado, A Body Corporate. Invention is credited to John F. Carpenter, Amber Haynes Fradkin, Theodore W. Randolph, Matthew B. Seefeldt.
Application Number | 20160002285 14/187055 |
Document ID | / |
Family ID | 45497408 |
Filed Date | 2016-01-07 |
United States Patent
Application |
20160002285 |
Kind Code |
A1 |
Seefeldt; Matthew B. ; et
al. |
January 7, 2016 |
THERAPEUTIC PROTEIN COMPOSITIONS HAVING REDUCED IMMUNOGENICITY
AND/OR IMPROVED EFFICACY
Abstract
The present invention provides methods for reducing and/or
evaluating the immunogenic potential of a therapeutic protein
preparation. The present invention further provides pharmaceutical
compositions of therapeutic proteins and methods of treatment with
the same, the compositions having low immunogenic potential and/or
improved efficacy. The invention achieves these goals by evaluating
therapeutic protein preparations for subvisible protein
particulates, which can contribute significantly to the overall
immunogenic potential of the protein preparation. Further, by
maintaining the content of such subvisible protein particulates to
below an immunogenic threshold level, the resulting pharmaceutical
composition is less likely to result in a loss of tolerance (e.g.,
upon repeated administration), thereby improving both the safety
and efficacy profile of the therapeutic.
Inventors: |
Seefeldt; Matthew B.;
(Boulder, CO) ; Randolph; Theodore W.; (Niwot,
CO) ; Fradkin; Amber Haynes; (Golden, CO) ;
Carpenter; John F.; (Denver, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Barofold, Inc.
The Regents of the University of Colorado, A Body
Corporate |
Aurora
Denver |
CO
CO |
US
US |
|
|
Assignee: |
Barofold, Inc.
Aurora
CO
The Regents of the University of Colorado, A Body
Corporate
Denver
CO
|
Family ID: |
45497408 |
Appl. No.: |
14/187055 |
Filed: |
February 21, 2014 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13186169 |
Jul 19, 2011 |
8697848 |
|
|
14187055 |
|
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61365728 |
Jul 19, 2010 |
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Current U.S.
Class: |
424/85.6 ;
424/134.1; 514/1.1; 514/11.3; 514/11.4; 530/350; 530/351;
530/387.3; 530/397 |
Current CPC
Class: |
C07K 2317/21 20130101;
C07K 16/241 20130101; C07K 16/26 20130101; C07K 14/565 20130101;
A61K 38/215 20130101; C07K 1/02 20130101; C07K 2317/14 20130101;
C07K 16/00 20130101; C07K 14/61 20130101; C07K 1/14 20130101; A61K
38/27 20130101 |
International
Class: |
C07K 1/02 20060101
C07K001/02; C07K 14/61 20060101 C07K014/61; C07K 14/565 20060101
C07K014/565; C07K 16/24 20060101 C07K016/24 |
Goverment Interests
STATEMENT OF GOVERNMENT SUPPORT
[0002] This invention was made with government support under grant
number R01-EB006006 awarded by the National Institutes of Health.
The government has certain rights in the invention.
Claims
1. A method for reducing immunogenicity of a therapeutic protein,
comprising: treating a therapeutic protein preparation under
conditions sufficient to reduce the amount of subvisible
particulates in the range of about 0.1 to about 10 microns in
size.
2. The method of claim 1, wherein the conditions comprise high
pressure treatment.
3. The method of claim 2, wherein the high pressure treatment is
configured to reduce the amount of the subvisible particulates, as
well as the amount of aggregates of greater than 10 microns in
size, and/or the amount of aggregates of less than about 0.1
microns in size.
4. The method of claim 1, wherein the conditions are selected to
favor and/or preserve monomeric protein.
5-9. (canceled)
10. The method of claim 1, wherein the therapeutic protein
preparation prior high pressure treatment is greater than 90%, or
greater than 95%, or greater than 97% monomeric material as
determined by size exclusion chromatography.
11. (canceled)
12. A pharmaceutical composition comprising a therapeutic protein
prepared by the process of claim 1.
13. A method for formulating a therapeutic protein, comprising:
reducing the amount of subvisible particulates in a therapeutic
protein preparation to below an immunogenic threshold level, the
subvisible particulates having a size in the range of about 0.1 to
about 10 microns.
14. The method of claim 13, wherein the amount of subvisible
particulates are reduced by high pressure treatment.
15. The method of claim 14, wherein the high pressure treatment is
configured to reduce the amount of the subvisible particulates, as
well as the amount of aggregates of greater than 10 microns in
size, and/or the amount of aggregates of less than about 0.1
microns in size.
16. The method of claim 14, wherein the high pressure treatment
favors and/or preserves monomeric protein.
17-18. (canceled)
19. The method of claim 16, wherein the therapeutic protein
preparation prior to treatment is greater than 90%, or greater than
95%, or greater than 97% monomeric material as determined by size
exclusion chromatography.
20-22. (canceled)
23. A therapeutic protein formulation prepared by the process of
claim 13.
24. A pharmaceutical composition comprising a therapeutic protein,
wherein subvisible particulates of the therapeutic protein having a
size in the range of about 0.1 to about 10 microns, are present
below an immunogenic threshold level.
25-26. (canceled)
27. A method for treating a disorder or disease in an animal,
comprising administering the pharmaceutical composition of claim 24
to said animal.
28-30. (canceled)
31. The method of claim 24, wherein the therapeutic protein
composition is greater than 90%, or greater than 95%, or greater
than 97% monomeric material as determined by size exclusion
chromatography.
32. The method of claim 31, wherein the therapeutic protein
composition is substantially chromatographically pure as determined
by SEC.
33. (canceled)
34. A method for producing an etanercept preparation with reduced
aggregate content, comprising: subjecting an etanercept preparation
to high pressure, at conditions sufficient for reducing particulate
content in the range of 0.1 to 10 microns in size.
35. (canceled)
36. The method of claim 34, wherein the conditions include high
pressure in the range of about 1000 bar to about 2500 bar, or from
1000 bar to about 2000 bar.
37. The method of claim 34, wherein the etanercept preparation
prior high pressure treatment is greater than 90%, or greater than
95%, or greater than 97% monomeric material as determined by size
exclusion chromatography.
38. (canceled)
Description
PRIORITY
[0001] This application claims the benefit of U.S. Provisional
Application No. 61/365,728, filed Jul. 19, 2010, which is hereby
incorporated by reference in its entirety.
BACKGROUND
[0003] Recombinant therapeutic proteins have had a significant
impact on the clinical treatment of diseases, including cancer,
over the past few decades. There are over 630 recombinant proteins
and peptides in commercial development, and protein-derived
therapeutics continue to grow rapidly relative to small molecule
therapeutics. As more recombinant proteins enter the pharmaceutical
market, the potential risks associated with these products are
becoming more of a concern. In particular, therapeutic proteins,
unlike small molecules, may be unstable and prone to aggregation
(Chi et al., Physical stability of proteins in aqueous solution:
Mechanism and driving forces in nonnative protein aggregation,
Pharmaceutical Research 20(9):1325-1336 (2003). Protein aggregation
can compromise the safety and effectiveness of the product.
[0004] Even though industry and regulatory agencies are aware of
aggregation and have policies and guidelines for their detection in
therapeutic protein compositions, some aggregates still go
undetected, in-part due to limitations of the conventionally
accepted analytical techniques. For instance, the USP currently has
no guidelines for detection of particles 0.1 to 10 microns in size.
Protein aggregates less than 0.1 micron are detected by analytical
methods such as size exclusion chromatography, and particles
greater than 10 microns are detected by the USP light obscuration
<788> technique. There are no clear recommendations for
detection of particles greater than about 0.1 micron but less than
about 10 microns, and the significance of these particles to the
immunogenic potential of the product has not been demonstrated.
This gap in subvisible particle detection leaves an opportunity for
protein aggregates to exist in approved commercial products and
current biologics undergoing development.
[0005] Protein aggregation occurs due to colloidal or conformation
instability allowing proteins to assemble with concomitant loss of
native structure and activity. Stresses such as freeze-thawing,
agitation (e.g. air-water interface), and UV light exposure, are
commonly encountered during processing, shipping, and storage of a
therapeutic product and are known to aggregate proteins (Chi et al.
2003). Aggregates may also be generated during protein purification
as the protein moves through a variety of solution exchanges at
high protein concentrations on column surfaces. Protein aggregation
may proceed through specific pathways that are initiated by
instability of the native protein conformation or colloid
instability associated with protein-protein interactions.
Conditions such as temperature, solution pH, ligands and cosolutes,
salt type and concentration, preservatives, and surfactants all
modulate protein structure and protein-protein interactions, and
thus aggregation propensity.
[0006] Aggregates produced as a result of different stresses may
exhibit different size distributions and their component proteins
may contain different secondary and tertiary structures, which
presumably expose different epitopes, potentially provoking immune
responses (Seefeldt et al., High-pressure studies of aggregation of
recombinant human interleukin-1 receptor antagonist:
thermodynamics, kinetics, and application to accelerated
formulation studies, Protein Sci. 14(9):2258-66 (2005)).
[0007] Protein aggregates present in therapeutic protein
compositions may not be recognized as "natural" by the immune
system. This might be due to exposure of a new epitope in the
aggregated protein that is not exposed in the non-aggregated
protein, or by formation in the aggregate of a new epitope, with
the result that the immune system is sensitized to the administered
recombinant protein aggregate. While in some instances the immune
system produces antibodies to the aggregates that do not neutralize
the therapeutic effect of the protein, in other cases, antibodies
are produced that bind to the recombinant protein and interfere
with the therapeutic activity, thereby resulting in declining
efficacy of the therapy.
[0008] Repeated administration of a recombinant protein can cause
acute and chronic immunologic reactions (Schellekens, H., Nephrol.
Dial. Transplant. 18:1257 (2003); Schellekens, H., Nephrol. Dial.
Transplant. 20 [Suppl 6]:vi3-vi9 (2005); Purohit et al. J. Pharm.
Sci. 95:358 (2006)). This loss or "breaking" of tolerance can have
serious effects including the development of autoimmune diseases.
For example, upon repeated administration of a recombinant protein,
tolerance can be broken, and an immune response produced against
the recombinant protein may cross-react with the individual's
endogenous protein. A mechanism for breaking self-tolerance was
demonstrated in transgenic mice immune tolerant for human
interferon-alpha 2. When preparations containing aggregates of
recombinant human interferon-alpha 2b were administered to the
mice, the mice lost tolerance for interferon-alpha 2 in a
dose-dependent manner (see Hermeling et al., J Pharm Sci. 95:1084
(2006)).
[0009] A loss of tolerance to an endogenously produced protein was
observed in patients using a preparation of recombinant
erythropoietin. Certain preparations of erythropoietin sold under
the trademark EPREX (Johnson & Johnson, New Brunswick, N.J.) in
Europe were found to break the immune tolerance of patients for
their own endogenous erythropoietin, leading to antibody-mediated
pure red cell aplasia (PRCA). The exogenous erythropoietin
preparation, which was administered to correct a deficiency in red
blood cell production, elicited the patient's immune system to
produce antibodies that neutralized endogenously produced
erythropoietin, causing a complete block in differentiation of red
blood cells. The cause of the immune response has been attributed
to leachates in the preparation which formed adjuvants with
erythropoietin (Boven et al., Nephrol. Dial. Transplant. 20 Suppl
3:iii33 (2005)), although other factors, such as aggregates, may
also be involved (Schellekens and Jiskoot, Nature Biotech. 24:613
(2006)).
[0010] Accordingly, new protein engineering and manufacturing
strategies are needed to minimize immunogenicity of protein
therapeutics and improve the effectiveness of therapy.
SUMMARY OF THE INVENTION
[0011] The present invention provides methods for reducing and/or
evaluating the immunogenic potential of a therapeutic protein
preparation. The present invention further provides pharmaceutical
compositions of therapeutic proteins and methods of treatment with
the same, the compositions having low immunogenic potential and/or
improved efficacy. The invention achieves these goals by evaluating
therapeutic protein preparations for subvisible particulates, which
can contribute significantly to the overall immunogenic potential
of the protein preparation. Further, by maintaining the content of
such subvisible particulates below an immunogenic level, the
resulting pharmaceutical composition is less likely to result in a
loss of tolerance (e.g., upon repeated administration), thereby
improving both the safety and efficacy profile of the
therapeutic.
[0012] Thus, in one aspect, the invention provides a method for
evaluating a therapeutic protein preparation for its immunogenic
potential. The invention comprises the use of micro-flow imaging
(MFI), or other methodology such as laser diffraction and/or
coulter counter, to evaluate particle numbers, sizes, and/or shape
in protein samples, and particularly in the subvisible range (e.g.,
about 0.1 to about 50 microns, or 0.1 to about 10 microns in size).
The presence and/or level of such subvisible particles is
indicative of an immunogenic preparation.
[0013] In accordance with the invention, the therapeutic may be a
recombinant protein preparation, and may comprise a monoclonal
antibody (which may be chimeric or humanized), an antigen binding
domain or single chain antibody, an Fc-domain containing protein
(e.g., ENBREL), or other therapeutic protein. Exemplary therapeutic
proteins are described herein, and include an interleukin or
interferon (e.g., an interferon-alpha, interferon-beta, or
interferon-gamma), protein or peptide hormone or growth factor
(e.g., insulin, GLP, erythropoietin, GM-CSF, or human growth
hormone), clotting factor (e.g., Factor VII, Factor VIII), or
enzyme for replacement therapy (e.g., uricase, MYOZYME,
phenylalanine hydroxylase, or phenylalanine ammonia lyase). The
protein may be produced recombinantly in E. coli, yeast, or
mammalian expression system (e.g., CHO cells), and at laboratory
scale or manufacturing scale. The protein may be recovered from
cells in soluble form, or recovered in insoluble form (e.g.,
inclusion bodies) and solubilized for evaluation.
[0014] The protein preparation, e.g., prior to evaluation and/or
treatment to reduce immunogenicity as described herein, may be
substantially free of visible aggregates as determined by light
obscuration for example, and/or may be substantially free of small
subvisible particulates of less than about 0.1 microns in size as
determined by, for example, size exclusion chromatography. The
protein preparation may be greater than about 90%, or about 95%, or
about 99% monomeric protein. In some embodiments, the protein
preparation is substantially chromatographically pure. In this
context, "substantially chromatographically pure" means that the
protein preparation does not contain detectable aggregates by SEC
analysis, or contains less than 1% aggregates by weight of protein
by SEC analysis.
[0015] In another aspect, the invention provides a method for
reducing the immunogenicity of a protein therapeutic, and/or
formulating a protein therapeutic so as to have low immunogenic
potential. The method comprises reducing the amount of particulates
in a subvisible range (e.g., about 0.1 to about 50 microns, or
about 0.1 to about 10 microns in size). In certain embodiments, the
level of such particulates is reduced by high pressure treatment of
the protein preparation as described in detail herein. The
conditions and/or parameters for high pressure treatment may be
selected and/or guided by MFI as well as other techniques disclosed
herein, so as to effectively reduce or eliminate the subvisible
particulates from the preparation, while favoring properly folded
monomeric protein. As disclosed herein, MFI analysis showed
particulate aggregates in commercial formulations that were not
detectable by SEC or visual inspection, and these solutions were
found to be immunogenic in mice. Particulate aggregate doses as low
as 1.6 ng/dose broke tolerance in mice and induced immune responses
to monomeric protein. When the preparation was treated with high
hydrostatic pressure the particulates were reduced to a dose level
of 0.02 ng/dose and the immunogenicity was eliminated.
[0016] As disclosed herein, a chromatographically pure mGH
preparation, which would conventionally be considered aggregate
free (and consequently the immunogenicity of the product would
conventionally be associated with something other than
aggregation), has aggregates present that cannot be detected by
chromatography, but are detectable by MFI. As shown herein, these
subvisible particulates are immunogenic since, by using high
pressure (for example), the subvisible particle content can be
decreased along with immunogenicity.
[0017] Further, and as disclosed herein, if conventional
pressure-treatment (e.g., as guided by SEC analysis) as described
in the art is applied to a therapeutic protein preparation such as
Enbrel, no change in aggregate level would be detected from the
treatment. However, by employing MFI, a different class of
aggregates is observed, subvisible particles, that may be reduced
via pressure, in a specific pressure window.
[0018] Further still, as shown herein, Betaseron also contains
subvisible particulates, which was previously unknown.
[0019] Thus, in still other aspects, the invention provides
pharmaceutical compositions and formulations comprising a
therapeutic protein, as well as methods of treatment with the same.
The composition contains subvisible (e.g., protein) particulates
(e.g., in the range of 0.1 to about 10 microns in size) at below an
immunogenic dose. The pharmaceutical composition may be formulated
for administration in a manner that, conventionally, has a tendency
to induce immune reactions to the therapeutic agent, such as
intra-muscular, subcutaneous, or intravenous administration. The
pharmaceutical composition in accordance with the invention has low
immunogenic potential (even for repeated and/or chronic treatment
regimens), may have a better safety and efficacy profile, as well
as better shelf stability.
[0020] Other objects and aspects of the invention will be apparent
from the following detailed description and the appended
claims.
DESCRIPTION OF THE FIGURES
[0021] FIG. 1A shows detection of aggregates in Product A
formulation, including after freeze-thaw (FT) and agitation
stresses.
[0022] FIG. 1B shows detection of aggregates in Product B
formulation, including after freeze-thaw (FT) and agitation
stresses.
[0023] FIG. 2A shows an increase in monomer content of Product A
formulation after high pressure treatment.
[0024] FIG. 2B shows an increase in monomer content of Product B
formulation after high pressure treatment.
[0025] FIG. 3 shows monomer and aggregate levels for samples used
in transgenic models.
[0026] FIG. 4 shows retention of native .alpha.-helical content of
aggregates produced by agitation and freeze-thawing, as determined
by infrared spectroscopy.
[0027] FIG. 5 shows retention of more .alpha.-helical content and
.beta.-sheet content in freeze-thaw aggregates, as compared to
agitated aggregates of Product B formulations, as determined by CD
spectroscopy.
[0028] FIG. 6 shows that, in naive and neonatally-primed mice,
maximum levels of hGH antibodies were observed in serum samples
collected in week 4.
[0029] FIG. 7 shows the results of high pressure treatment on
immunogenicity of Product A and Product B samples in the
neonatally-primed mouse model.
[0030] FIG. 8 shows the results of high pressure treatment on
immunogenicity of Product A and Product B samples in the naive
adult mouse model.
[0031] FIG. 9 shows that no immune responses were observed in the
transgenic mouse model.
[0032] FIG. 10A shows the number counts of particles 1-50 .mu.m in
size in mGH stock preparation before and after high pressure
treatment, represented by the black and grey bars respectively.
[0033] FIG. 10B shows the number counts of particles 1-50 .mu.m in
size in mGH agitated preparation before and after high pressure
treatment, represented by the black and grey bars respectively.
[0034] FIG. 10C shows the number counts of particles 1-50 .mu.m in
size in mGH freeze-thaw preparation before and after high pressure
treatment, represented by the black and grey bars respectively.
[0035] FIG. 11A shows the number of particles 1-50 .mu.m in size
for mGH adsorbed to alum.
[0036] FIG. 11B shows the number of particles 1-50 .mu.m in size
for mGH adsorbed to glass.
[0037] FIG. 12 shows the second derivative infrared spectroscopy of
aggregated mGH preparations. The mGH adsorbed to alum and glass
microparticles are represented by solid black and dotted grey
lines, respectively. The mGH aggregates produced in freeze-thaw and
agitation are shown as dark grey dashed and grey dotted-dashed
lines, respectively.
[0038] FIG. 13 Stern-Volmer plot of native, unfolded and particle
adsorbed mGH. The native and unfolded protein solutions are
represented by white circles and white triangles respectively.
Protein adsorbed to Alhydrogel and glass particles are shown as
white diamonds and white squares respectively. Some error bars are
smaller than data point symbols.
[0039] FIG. 14 shows IgG1 antibody production for each mGH
preparation. Antibody responses from bleeds from days 21, 35, 42
and 49 are represented from left to right as black, horizontal
lined, diagonal lined, and crosshatched bars respectively. Only
positive mice were averaged. Error bars represent 95% confidence
intervals. Numbers above bars indicate number of positive mice.
[0040] FIG. 15A shows IgG2a antibody production for each mGH
preparation. Antibody responses from bleeds from days 21, 35, 42
and 49 are represented from left to right as black, horizontal
lined, diagonal lined, and crosshatched bars respectively. Only
positive mice were averaged. Error bars represent 95% confidence
intervals. Numbers above bars indicate number of positive mice.
[0041] FIG. 15B shows IgG2b antibody production for each mGH
preparation. Antibody responses from bleeds from days 21, 35, 42
and 49 are represented from left to right as black, horizontal
lined, diagonal lined, and crosshatched bars respectively. Only
positive mice were averaged. Error bars represent 95% confidence
intervals. Numbers above bars indicate number of positive mice.
[0042] FIG. 15C shows IgG2c antibody production for each mGH
preparation. Antibody responses from bleeds from days 21, 35, 42
and 49 are represented from left to right as black, horizontal
lined, diagonal lined, and crosshatched bars respectively. Only
positive mice were averaged. Error bars represent 95% confidence
intervals. Numbers above bars indicate number of positive mice.
[0043] FIG. 16 shows IgG3 antibody production for each mGH
preparation. Antibody responses from bleeds from days 21, 35, 42
and 49 are represented from left to right as black, horizontal
lined, diagonal lined, and crosshatched bars respectively. Only
positive mice were averaged. Error bars represent 95% confidence
intervals. Numbers above bars indicate number of positive mice.
[0044] FIG. 17 shows MFI total particle analysis of Betaseron.
[0045] FIG. 18 shows MFI particle size distribution analysis of
Betaseron. To get an approximate mass of protein in the subvisible
particles present in Betaseron formulation the mass of each
particle was determined by assuming a density of the spherical
particles of 1.2 g/ml. An approximation of the mass percentage of
total protein forming subvisible particles is 2.36%+/-0.83%.
[0046] FIG. 19 shows Log 10 relative serum antibody potency of
binding antibodies to BaroFeron, Avonix, and Betaseron. BaroFeron
data is shown in red, Avonix in green, and Betaseron in Blue.
Dosing of Betaseron resulted in a significant development of
binding antibodies to monomeric Betaseron relative to baseline.
Neither Avonix or BaroFeron developed a significant response.
[0047] FIG. 20A shows that Etanercept has aggregates in its final
formulation. Etanercept was diluted 5.times. in formulation buffer
and analyzed by SEC-HPLC.
[0048] FIG. 20B shows that Etanercept has aggregates in its final
formulation. Etanercept was diluted 5.times. in formulation buffer
and analyzed by MFI.
[0049] FIG. 21 shows Etanercept diluted to 10 mg/ml, pressure
treated, and analyzed by SEC-HPLC. No difference between
atmospheric and pressure treated samples can be detected with this
analytical method.
[0050] FIG. 22 shows that pressure decreases particles by >25%
in the range of 1000-2000 bar. Etanercept was diluted to 10 mg/ml,
pressure treated and analyzed by MFI.
[0051] FIG. 23A shows that pressure of 2000 bar decreases
subvisible particles by >30% in Etanercept formulations.
Etanercept was treated in quadruple at 2000 bar and analyzed by
MFI. Data for all samples are shown.
[0052] FIG. 23B shows that pressure of 2000 bar decreases
subvisible particles by >30% in Etanercept formulations.
Etanercept was treated in quadruple at 2000 bar and analyzed by
MFI. Data for the average of treatment at 2000 bar and atmospheric
are shown.
DETAILED DESCRIPTION OF THE INVENTION
[0053] The present invention provides methods for reducing and/or
evaluating the immunogenic potential of a therapeutic protein
preparation. The present invention further provides pharmaceutical
compositions and formulations of therapeutic proteins, and methods
of treatment with the same, the compositions and formulations
having low immunogenic potential and/or improved efficacy.
[0054] A "therapeutic protein preparation" is any composition
comprising a protein for human or veterinary therapy. The
preparation is generally a liquid composition comprising soluble
protein, and thus may also comprise one or more pharmaceutically
acceptable diluents and/or excipients, such as water, buffer, a
pharmaceutically acceptable carrier, and/or a denaturant solution.
Components that are not pharmaceutically acceptable, but which are
useful in the manufacturing and purification of recombinant
protein, may also be present. Such components are removed from the
commercial formulation.
[0055] The therapeutic protein may be intended for acute or chronic
administration, such as, for example, approximately daily, weekly,
or monthly administration for a period of months (e.g., at least 6
months) or even years (e.g., 1, 2, or 3 or more years). For
example, the therapeutic protein may be indicated for treatment of
a chronic disease, such as diabetes mellitus, chronic viral
infection (e.g., hepatitis), asthma, COPD, or an autoimmune
disorder, such as multiple sclerosis (or other demyelinating
disorder) or rheumatoid arthritis, or clotting or enzyme
deficiency. The therapeutic protein may be indicated for the
treatment of cancer. By providing therapeutic protein compositions
having low immunogenic potential, the invention allows prolonged
therapy without breaking tolerance and/or without diminishing
therapeutic effect.
[0056] The protein preparation may be a laboratory sample, bulk
pharmaceutical preparation, or individual dosage unit. In some
embodiments, the protein preparation is supplied in a formulation
suitable for administration, to evaluate its immunogenic potential
and/or to further reduce its immunogenic potential as described
herein. Alternatively, the therapeutic protein preparation may be
at a larger laboratory scale or manufacturing scale (e.g., 10 L,
100 L, 1000 L, 10,000 L, 20,000 L or more), for reduction in its
immunogenic potential by removal (or monomerization) of soluble
protein aggregates of visible and subvisible sizes (e.g., including
subvisible particulates in the 0.1 to 10 micron range), and/or
other non-native proteins. The therapeutic protein preparation can
then be adjusted to comprise a formulation suitable for
administration as a drug with low immunogenic potential.
[0057] The protein may be a recombinant protein therapeutic, such
as an immunoglobulin (e.g, a monoclonal antibody, which may be
chimeric or humanized), an antigen-binding domain or single chain
antibody, an Fc-domain containing protein (e.g., ENBREL), or other
therapeutic protein. Where the protein comprises an antibody or
antibody domain, the antibody or domain may be of any human
isotype, such as an IgG isotype. Exemplary therapeutic proteins
include a an interleukin or interferon (e.g., an interferon-alpha,
interferon-beta, or interferon-gamma), protein or peptide hormone
or growth factor (e.g., insulin, GLP, erythropoietin, GM-CSF, or
human growth hormone), clotting factor (e.g., Factor VII, Factor
VIII), or enzyme for replacement therapy (e.g., uricase, MYOZYME,
phenylalanine hydroxylase, phenylalanine ammonia lyase). The
therapeutic protein may comprise full length proteins, or
functional portions thereof, and may contain modifications known in
the art for enhancing activity and/or stability of the
molecule.
[0058] The recombinant therapeutic protein may be a large protein
of one or more than one subunit. For example, the protein may have
a size greater than about 500 kDa, 400 kDa, 200 kDa, 100 kDa, 75
kDa, 50 kDa, 40 kDa, 30 kDa, 20 kDa, 10 kDa, 5 kDa, 2 kDa, or 0.25
kDa. In certain embodiments, the recombinant protein comprises a
plurality of polypeptide chains, which may optionally be connected
by one or more disulfide bonds.
[0059] Exemplary therapeutic proteins include interferon-alpha;
interferon-alpha 2a (Roferon-A; Pegasys); interferon-beta Ib
(Betaseron); interferon-beta Ia (Avonex); insulin (e.g., Humulin-R,
Humalog); DNAase (Pulmozyme); Neupogen; Epogen; Procrit (Epotein
Alpha); Aranesp (2nd Generation Procrit); Intron A
(interferon-alpha 2b); Rituxan (Rituximab anti-CD20); IL-2
(Proleukin); IL-I ra (Kineret); BMP-7 (Osteogenin); TNF-alpha Ia
(Beromun); HUMIRA (anti-TNF-alpha MAB); tPA (Tenecteplase); PDGF
(Regranex); interferon-gamma Ib (Actimmune); uPA; GMCSF; Factor
VII, Factor VIII; Remicade (infliximab); Enbrel (Etanercapt);
Betaferon (interferon beta-Ia); Saizen (somatotropin); Erbitux
(cetuximab); Norditropin (somatropin); Nutropin (somatropin);
Genotropin (somatropin); Humatrope (somatropin); Rebif (interferon
beta Ia); Herceptin (trastuzumab); abatacept (Orencia) and Humira
(adalimumab); Xolair (omalizumab); Avastin (bevacizumab); Neulasta
(pegfilgrastin); Cerezyme (Imiglucerase); and motavizumab. The
amino acid sequence and/or structure of such therapeutic proteins
are known in the art, and such sequences/structures are hereby
incorporated by reference.
[0060] The therapeutic protein may contain one or a plurality of
glycosylations, or may comprise one or a plurality of PEG strands
covalently attached. In other embodiments, the protein therapeutic
is a recombinant fusion protein with a half-life extending fusion
partner (e.g., albumin or antibody Fc domain).
[0061] The protein may be produced recombinantly in an E. coli,
yeast (e.g., Pichia), mammalian cell system (e.g., CHO cells) or
other system, and at a manufacturing scale as described. The
protein may be recovered in soluble form to evaluate and/or reduce
its immunogenic potential as described herein. In other
embodiments, the protein preparation is recovered from cells in an
insoluble form (e.g., inclusion bodies or precipitate), and
subsequently solubilized for evaluating and/or reducing immunogenic
potential as described herein.
[0062] The protein preparation, prior to evaluation or treatment to
reduce immunogenic potential, may be substantially free of visible
aggregates as determined by, for example, light obscuration
techniques. Such visible aggregates may be of the size of about 50
microns or larger. The protein preparation may further be
substantially free of small soluble protein aggregates (e.g.,
subvisible aggregates) of less than about 0.2 or about 0.1 microns
in size, as determined, for example, by size exclusion
chromatography. In other embodiments, the preparation further
contains significant amounts of visible aggregates and/or small
soluble aggregates, whose level or concentration may be further
reduced in accordance with the invention. The protein preparation
may be greater than 90%, or 95%, or 97%, or 98%, or 99% monomeric
protein, or in some embodiments may be substantially
chromatographically pure as determined by SEC.
[0063] The protein preparation may contain an immunogenic amount of
subvisible particles in the 0.1 to 10 micron range, such as greater
than 2 ng/ml, greater than about 5 ng/ml, greater than about 10
ng/ml, greater than about 50 ng/ml, greater than about 100 ng/ml,
greater than about 200 ng/ml, or greater than about 500 ng/ml of
such subvisible particulates. The immunogenic potential of the
preparation may be determined and/or quantified by a method
described herein, including the ability of the protein preparation
to elicit antibodies in a suitable animal model and/or human
population.
[0064] In some embodiments, the protein preparation has been
resolubilized using chaotrope treatment. Proteins produced in
microbial systems are usually insoluble and thus require chaotrope
treatment for solubilization and renaturation. Typically, high
concentrations of chemicals (e.g., 6M GdnHCl or 8M urea) are
required to dissolve the aggregates. After protein dissolution, the
chaotrope is diluted to both decrease the protein concentration and
allow the protein molecules to return to their native conformation.
This process requires denaturation prior to refolding. Because
proteins tend to (re)aggregate during refolding, yields of properly
folded protein are never 100% in chaotrope-based processes.
Accordingly, the invention in certain embodiments, avoids the use
of chaotrope treatment.
[0065] In order to evaluate the protein preparation for immunogenic
potential, the invention employs micro-flow imaging (MFI) to
evaluate particle numbers and particle sizes of protein samples,
and particularly in the subvisible range (e.g., about 0.1 to about
10 microns in size). The presence and/or level of such subvisible
particles is indicative of an immunogenic preparation.
[0066] Alternatively, particles in the subvisible range may be
detected and/or quantified by laser diffraction or Coulter Counter.
The protein preparation may be evaluated by a coulter counter,
which can determine particle counts in the 400 nm to 1.7 .mu.m
range and is limited by the conductivity of the protein solution.
Static light scattering (laser diffraction) can be used to evaluate
particle content in 40 nm to 8 mm range, however this technique is
generally not considered quantitative and cannot count the number
of particles present or the size distribution of the particles.
[0067] Even though industry and regulatory agencies are aware of
aggregation and have policies and guidelines for their detection in
therapeutic protein compositions, some aggregates still go
undetected, in-part due to the conventionally accepted analytical
techniques. For instance, the USP currently has no guidelines for
detection of particles 0.1 to 10 microns in size. Protein
aggregates less than 0.1 micron are detected by analytical methods
such as size exclusion chromatography, and particles greater than
10 microns are detected by the USP light obscuration <788>
technique. There are no clear recommendations for detection of
particles greater than about 0.1 micron but less than about 10
microns. This gap in subvisible particle detection leaves an
opportunity for protein aggregates to exist in approved commercial
products.
[0068] More specifically, characterization of aggregates and
particulates in final formulations has previously been difficult
(Carpenter et al., Overlooking Subvisible Particles in Therapeutic
Protein Products: Gaps That May Compromise Product Quality, J.
Pharmaceutical Sciences 98:4 (2009). SEC-HPLC is the industry
standard due to its high throughput and relative robustness.
However, particles and aggregates greater than about 0.1 microns
can be filtered on the column head, preventing an accurate
assessment of all of the aggregates that are present in the
solution. More sophisticated methods such as analytical
ultracentrifugation can monitor aggregate content and size without
a column, however large particles settle too quickly and cannot be
quantified. The recent development of micro-flow imaging provides a
new technology for visibly measuring the particle content of a
solution, and can assess aggregates that previously have not been
identified or characterized. During micro-flow imaging, digital
microscopy images of a protein solution are taken relative to a
blank, and aggregate content is measured by quantifying the size
and number of particles present. An apparatus for micro-flow
imaging is commercially available from Brightwell Technologies,
Inc.
[0069] Protein preparations may further be characterized for
aggregate or particulate content by one or a plurality of
additional analytical techniques selected from the following.
[0070] The protein preparation may be evaluated by analytical
ultracentrifugation. The use of analytical ultracentrifugation for
characterization of aggregation of protein therapeutics is
discussed in Philo, J. S., American Biotechnology Laboratory, page
22, October 2003. Experiments that can be performed using
analytical ultracentrifugation include sedimentation velocity and
sedimentation equilibrium experiments, which can be performed to
determine whether multiple solutes exist in a solution (e.g.,
monomer, dimer, trimer, etc.) and provide an estimate of molecular
weights for the solutes.
[0071] The protein preparation may be evaluated by size-exclusion
chromatography and gel permeation chromatography, which can
estimate molecular weights and aggregation numbers of proteins.
Such techniques also separate out various protein aggregates. See
Wu, C-S. (editor), Handbook of Size Exclusion Chromatography and
Related Techniques, Second Edition (Chromatographic Science),
Marcel Dekker: New York, 2004 (particularly chapter 15 at pages
439-462 by Baker et al., "Size Exclusion Chromatography of
Proteins") and Wu, C-S. (editor), Column Handbook for Size
Exclusion Chromatography, San Diego: Academic Press, 1999
(particularly Chapters 2 and 18).
[0072] The protein preparation may be evaluated by field flow
fractionation, which relies on a field perpendicular to a liquid
stream of molecules. Field flow fractionation can be used to
analyze and separate aggregated proteins such as protein monomers,
dimers, trimers, etc. See Zhu et al., Anal. Chem. 77:4581 (2005);
Litzen et al., Anal. Biochem. 212:469 (1993); and Reschiglian et
al., Trends Biotechnol. 23:475 (2005).
[0073] The protein preparation may be evaluated by light scattering
methods, such as methods using laser light scattering (often in
conjunction with size-exclusion chromatography or other methods).
Light scattering methods can also be used to estimate the molecular
weight of proteins, including protein aggregates; see, for example,
Mogridge, J., Methods Mol Biol. 261:113 (2004) and Ye, H.,
Analytical Biochem. 356:76 (2006). Dynamic light scattering
techniques are discussed in Pecora, R., ed., Dynamic Light
Scattering: Applications of Photon Correlation Spectroscopy, New
York: Springer Verlag, 2003 and Berne, B J. and Pecora, R., Dynamic
Light Scattering: With Applications to Chemistry, Biology, and
Physics, Mineola, N.Y.: Dover Publications, 2000. Laser light
scattering is discussed in Johnson, C S. and Gabriel, D. A., Laser
Light Scattering, Mineola, N.Y.: Dover Publications, 1995, and
other light scattering techniques which can be applied to determine
protein aggregation are discussed in Kratochvil, P., Classical
Light Scattering from Polymer Solutions, Amsterdam: Elsevier,
1987.
[0074] Light obscuration can also be used to measure protein
aggregation of the preparation; see Seefeldt et al., Protein Sci.
14:2258 (2005); Kim et al., J. Biol. Chem. 276: 1626 (2001); and
Kim et al., J. Biol. Chem. 277: 27240 (2002).
[0075] The protein preparation may be evaluated by fluorescence
spectroscopy, such as fluorescence anisotropy spectroscopy, which
can be used to determine the presence of protein aggregates.
Fluorescence probes (dyes) can be covalently or non-covalently
bound to the aggregate to aid in analysis of aggregates (see, e.g.,
Lindgren et al., Biophys. J. 88: 4200 (2005)), US Patent
Application Publication 2003/0203403), or Royer, C. A., Methods
Mol. Biol. 40:65 (1995). Internal tryptophan residues can also be
used to detect protein aggregation by fluorescence; see, e.g., Dusa
et al., Biochemistry 45:2752 (2006).
[0076] Many methods of gel electrophoresis (e.g., denaturing or
non-denaturing PAGE) can be employed to analyze proteins and
protein aggregation. Native PAGE (non-denaturing PAGE) can be used
to study non-covalently linked aggregates. See, e.g., Hermeling et
al. J. Phar. Sci. 95:1084-1096 (2006); Kilic et al., Protein Sci.
12:1663 (2003); Westermeier, R., Electrophoresis in Practice: A
Guide to Methods and Applications of DNA and Protein Separations
4th edition, New York: John Wiley & Sons, 2005; and Hames, B.
D. (Ed.), Gel Electrophoresis of Proteins: A Practical Approach,
3rd edition, New York: Oxford University Press, USA, 1998.
[0077] The protein preparation may be evaluated by gas-phase
electrophoretic mobility molecular analysis (GEMMA) (see Bacher et
al., J. Mass Spectrom. 36:1038 (2001). A combination of
electrophoresis in the gas phase and mass spectrometry provides
another method of analyzing protein complexes and aggregates.
[0078] Nuclear magnetic resonance spectroscopic techniques can be
used to estimate hydrodynamic parameters related to protein
aggregation. See, for example, James, T. L. (ed.), Nuclear Magnetic
Resonance of Biological Macromolecules, Part C, Volume 394: Methods
in Enzymology, San Diego: Academic Press, 2005; James, T. L.,
Dotsch, V. and Schmitz, U. (eds.), Nuclear Magnetic Resonance of
Biological Macromolecules, Part A (Methods in Enzymology, Volume
338) and Nuclear Magnetic Resonance of Biological Macromolecules,
Part B (Methods in Enzymology, Volume 339), San Diego: Academic
Press, 2001, and Mansfield, S. L. et al., J. Phys. Chem. B,
103:2262 (1999). Linewidths, correlation times, and relaxation
times are among the parameters that can be measured to estimate
tumbling time in solution, which can then be correlated with the
state of protein aggregation. Electron paramagnetic resonance (EPR
or ESR) can also be used to determine aggregation states; see,
e.g., Squier et al., J. Biol. Chem. 263:9162 (1988).
[0079] As disclosed herein, subvisible protein particulates at
levels undetectable by standard analytical methods such as size
exclusion chromatography and light obscuration can induce immune
responses to a self protein or epitope. Specifically, aggregates
detected by MFI, which could not previously be detected by SEC-HPLC
as they were below the limit of detection, can have significant
immunogenic potential.
[0080] The present invention provides methods for evaluating
protein aggregation, including the level and concentration of
subvisible protein particulates in protein preparations. As used
herein, a "protein aggregate" or "protein particulate" is defined
as being composed of a multiplicity of protein molecules wherein
non-native noncovalent interactions and/or non-native covalent
bonds (such as non-native intermolecular disulfide bonds) hold the
protein molecules together. The aggregates may be soluble or
insoluble. Protein aggregates include, but are not limited to,
inclusion bodies, soluble and insoluble precipitates, soluble
non-native oligomers, gels, fibrils, films, filaments,
protofibrils, amyloid deposits, plaques, and dispersed non-native
intracellular oligomers.
[0081] The protein preparation, which may contain aggregates or
protein particulates, may be of high monomer content (for example,
at least 80% monomer; at least about 90% monomer; at least about
95% monomer; at least about 97% monomer, at least about 98%
monomer, or at least about 99% monomer). Such preparations of high
monomer content may still retain significant immunogenic potential
due to the presence of even relatively low amounts of subvisible
particulates in the range of about 0.1 to 10 microns. In some
embodiments, such subvisible particulates are greater than about
0.2 microns, greater than about 0.3 microns, or greater than about
0.4 or 0.5 microns. Such subvisible particulates may be less than
about 8 microns, less than about 5 microns, or less than about 3 or
2 microns.
[0082] Where such subvisible particulates, or other aggregate
content, is detectable via the techniques described herein, the
invention provides methods of reducing such particulate or
aggregate content.
[0083] Aggregates and particulates in some embodiments might be
removed by filtration, purification, and refolding. All protein
therapeutics are sterile-filtered prior to final formulation
(Carpenter, Randolph et al. 2009). However, membrane filtration is
not a benign process and exposes proteins to large amounts of
surface area (Maa and Hsu 1998). Many proteins are highly surface
active, and adsorption to interfaces can lead to protein
aggregation. Accordingly, filtration does not always provide a
viable option for aggregate removal. Further, with a filtration
cutoff of about 0.2 microns or greater, some particulates will
escape filtration.
[0084] Column-based protein purification processes are commonly
employed for aggregate and particulate removal. Unfortunately,
yields of process chromatography steps such as size exclusion-,
anionic-, or hydrophobic interaction chromatography are rarely 100%
as aggregated proteins will typically elute near the native
protein. Consequently, the manufacturer typically faces the choice
between having a suitable yield or low aggregate burden.
[0085] In some embodiments, particulate and/or aggregate content is
reduced in the preparation by subjecting the preparation to
high-pressure conditions. Generally, the high pressure conditions
are selected to not induce aggregation, where the conditions
include magnitude of high pressure, duration of high-pressure
treatment, protein concentration, temperature, pH, ionic strength,
chaotrope concentration, surfactant concentration, buffer
concentration, preferential excluding compounds concentration, or
other solution parameters as described herein. See WO 2008/033556,
which is hereby incorporated by reference in its entirety.
[0086] In some embodiments, particulate and/or aggregate content is
reduced with high pressure after purification of the protein is
completed, that is, after the protein is at the desired purity
level for use as a therapeutic (where purity refers to undesired
components besides the protein of interest, but not to aggregates
or particulate of the protein of interest).
[0087] For example, high pressure conditions may be selected to
favor properly folded monomeric protein, and to reduce protein
particulates (e.g., subvisible particulates) by at least 2 fold, 5
fold, 10 fold, 50 fold, 100 fold, or more. In some embodiments,
subvisible particulates are reduced to below a detectable level as
determined by MFI.
[0088] As used herein, the term "high pressure" means a pressure of
at least about 250 bar. The high pressure treatment in accordance
with embodiments of the invention may be at least about 250 bar of
pressure, at least about 400 bar of pressure, at least about 500
bar of pressure, at least about 1 kbar of pressure, at least about
2 kbar of pressure, at least about 3 kbar of pressure, at least
about 5 kbar of pressure, or at least about 10 kbar of pressure.
"Atmospheric," "ambient," or "standard" pressure is defined as
approximately 15 pounds per square inch (psi) or approximately 1
bar or approximately 100,000 Pascals.
[0089] Use of high pressure treatment to reduce subvisible
particulate and/or aggregate content in therapeutic protein
preparations may extend the shelf life of such preparations, such
that the immunogenic potential is reduced or eliminated for a
period of time. Thus, in accordance with certain embodiments, high
pressure treatment is performed at any time prior to use of the
pharmaceutical for human therapy, for example, at least about 3
years before the protein composition is intended to be administered
to a individual, at least about 2 years, at least about 1 year, at
least about 6 months, at least about 3 months, at least about 1
month, or at least about 2 weeks before the protein composition is
intended to be administered to a individual.
[0090] Conditions favorable to reduction or elimination of the
particulates and/or aggregates in a protein preparation with high
monomer content may not be the same or similar to the conditions
favorable to maximum yield of protein recovery from a highly
aggregated solution. This distinction arises from the common
observation that pressure treatment in many solution conditions can
induce aggregation and particulate formation of monomeric species
(Ferrao-Gonzales, et al. (2000), PNAS 97(12):6445-6450; Kim, et al.
(2002), Journal of Biological Chemistry 277(30):27240-27246;
Seefeldt, et al. (2005), Protein Science 14(9): 2258-2266; Dzwolak,
W. (2006), Biochimica Et Biophysica Acta-Proteins And Proteomics
1764(3): 470-480; Grudzielanek, et al. (2006), Journal Of Molecular
Biology 356(2): 497-509; Kim, et al. (2006), High-pressure studies
on protein aggregates and amyloid fibrils. Amyloid, Prions, And
Other Protein Aggregates, Pt C. 413: 237-253). The selection of
such conditions for high pressure should thus be guided by MFI, as
well as other techniques, including SE chromatography, light
scattering, and/or CD spectroscopy, among others.
[0091] High pressure treatment provides an effective process for
the removal of protein particulates (including subvisible
particulates) and aggregates because it does not involve filtration
or purification, which tend to induce aggregation. However,
conditions must be identified that do not induce aggregation of the
monomer (in any form) while still dissociating aggregates and
particulates. High pressure refolding has been identified to occur
at conditions within a "pressure-window" that generally favors the
native protein conformation. For example, as shown herein, in some
embodiments the pressure window for reduction of subvisible
particulates may be from about 1000 bar to about 2500 bar, or about
1000 bar to about 2000 bar. In other embodiments, the window may be
about 1250 bar to about 2250 bar, or about 1500 bar to about 2000
bar.
[0092] In some embodiments, high-pressure treatment is conducted
after filtration. While filtration may remove large protein
aggregates, as well as particulates above about 0.2 or 0.5 microns
in size, subsequent high pressure treatment can reduce the level of
particles in the subvisible range that may be induced by the
filtration process itself, or which may escape filtration.
[0093] Several conditions can be adjusted for optimal treatment of
the protein preparation to reduce particulates and aggregates that
may result in immunogenicity. Proteins can be treated by high
pressure by placing them in a vessel (which can be a high-pressure
variable-volume loading device) and then placing the vessel in a
high-pressure generator, such as those available from High Pressure
Equipment Co., Erie, Pa. High-pressure techniques are described in
U.S. Pat. Nos. 6,489,450 and 7,064,192, U.S. Patent Application
Publication No. 2004/0038333, and International Patent Application
WO 02/062827; the methods for generating high pressure described
therein are hereby incorporated by reference herein in their
entirety. Certain devices have also been developed which are
particularly suitable for refolding of proteins under high
pressure; see International Patent Application Publication No. WO
2007/062174, which is hereby incorporated by reference in its
entirety. Condition parameters to be adjusted for favorable high
pressure treatment are described below.
[0094] The concentration of protein may be adjusted for optimal
reduction of subvisible particulates. Protein concentrations of at
least about 0.1 mg/ml, at least about 1.0 mg/ml, at least about 5.0
mg/ml, at least about 10 mg/ml, or at least about 20 mg/ml may be
used. Generally, the protein will be present in a concentration of
from about 0.01 or about 0.1 mg/ml to about 50, 250, or 400
mg/ml.
[0095] The duration of high pressure treatment may be selected for
reduction of subvisible particulates. Generally, high pressure
treatment may be conducted for about 15 minutes to about 50 hours,
or possibly longer. In some embodiments, the duration of high
pressure treatment is up to about 1 week, about 5 days, about 4
days, about 3 days, etc.). Thus, in some embodiments, the duration
sufficient to reduce the level of subvisible particulates is from
about 2 to about 30 hours, from about 2 to about 24 hours, from
about 2 to about 18 hours, or from about 1 to about 10 hours.
[0096] The protein preparation may be in aqueous solution
conditions to favor properly folder monomeric protein, and to
reduce subvisible particulates by high pressure. The solution
components may be one or more agents selected from one or more
stabilizing agents, one or more buffering agents, one or more
surfactants, one or more disulfide shuffling agent pairs, one or
more salts, one or more chaotropes, or combinations of two or more
of the foregoing. Where such component(s) are not pharmaceutically
acceptable, the added component(s) should be removable from the
protein preparation prior to administration as a pharmaceutical.
Such components may be removed by dialysis.
[0097] Exemplary agents include, but are not limited to, buffers
(examples include, but are not limited to, phosphate buffer, borate
buffer, carbonate buffer, citrate buffer, HEPES, MEPS), salts
(examples include, but are not limited to, the chloride, sulfate,
and carbonate salts of sodium, zinc, calcium, ammonium and
potassium), chaotropes (examples include, but are not limited to,
urea, guanidine hydrochloride, guanidine sulfate and sarcosine),
and stabilizing agents (e.g., preferential excluding compounds,
etc.).
[0098] Non-specific protein stabilizing agents act to favor the
most compact conformation of a protein. Such agents include, but
are not limited to, one or more free amino acids, one or more
preferentially excluding compounds, trimethylamine oxide,
cyclodextrans, molecular chaperones, and combinations of two or
more of the foregoing.
[0099] Amino acids can be used to prevent reaggregation and
facilitate the dissociation of hydrogen bonds. Typical amino acids
that can be used, without limitation, are arginine, lysine,
proline, glycine, histidine, and glutamine or combinations of two
or more of the foregoing. In some embodiments, the free amino
acid(s) is present in a concentration of about 0.1 mM to about the
solubility limit of the amino acid, and in some variations from
about 0.1 mM to about 2 M. The optimal concentration is a function
of the desired protein and should favor the native
conformation.
[0100] Preferentially excluding compounds can be used to stabilize
the native conformation of the protein of interest. Possible
preferentially excluding compounds include, but are not limited to,
sucrose, hexylene glycol, sugars (e.g., sucrose, trehalose,
dextrose, mannose), and glycerol. The range of concentrations that
can be use are from 0.1 mM to the maximum concentration at the
solubility limit of the specific compound. Exemplary concentrations
include those that are consistent with physiological osmolality.
The optimum preferential excluding concentration is a function of
the protein of interest.
[0101] In some embodiments, a stabilizing agent is employed, such
as one or more of sucrose, trehalose, glycerol, betaine, amino
acid(s), or trimethylamine oxide. In certain embodiments, the
stabilizing agent is a cyclodextran. In some embodiments, the
cyclodextran is present in a concentration of about 0.1 mM to about
the solubility limit of the cyclodextran. In certain embodiments,
the stabilizing agent is a molecular chaperone.
[0102] A single stabilizing agent maybe be used or a combination of
two or more stabilizing agents (e.g., at least two, at least three,
or 2 or 3 or 4 stabilizing agents). Where more than one stabilizing
agent is used, the stabilizing agents may be of different types,
for example, at least one preferentially excluding compound and at
least one free amino acid, at least one preferentially excluding
compound and betaine, etc.
[0103] Buffering agents may be present to maintain a desired pH
value or pH range. Numerous suitable buffering agents are known to
the skilled artisan and should be selected based on the pH that
favors (or at least does not disfavor) the native (monomeric)
conformation of the protein of interest. Either inorganic or
organic buffering agents may be used.
[0104] Thus, in some embodiments, at least one inorganic buffering
agent is used (e.g., phosphate, carbonate, etc.). In certain
embodiments, at least one organic buffering agent is used (e.g.,
citrate, acetate, Tris, MOPS, MES, HEPES, etc.). Additional organic
and inorganic buffering agents are well known to the art.
[0105] A surfactant, a surface active compound, may also be
employed to reduce the surface tension of the water. Surfactants
may also improve the solubility of the protein of interest.
Surfactants may be used at concentrations above or below their
critical micelle concentration (CMC), for example, from about 5% to
about 20% above or below the CMC. However, these values will vary
dependent upon the surfactant chosen, for example, surfactants such
as, beta-octylgluco-pyranoside may be effective at lower
concentrations than, for example, surfactants such as TWEEN-20
(polysorbate 20). The optimal concentration is a function of each
surfactant, which has its own CMC.
[0106] Useful surfactants include nonionic (including, but not
limited to, t-octylphenoxypolyethoxy-ethanol and polyoxyethylene
sorbitan), anionic (e.g., sodium dodecyl sulfate) and cationic
(e.g., cetylpyridinium chloride) and amphoteric agents. Suitable
surfactants include, but are not limited to deoxycholate, sodium
octyl sulfate, sodium tetradecyl sulfate, polyoxyethylene ethers,
sodium cholate, octylthioglucopyranoside, n-octylglucopyranoside,
alkyltrimethylanmonium bromides, alkyltrimethyl ammonium chlorides,
non-detergent sulfobetaines, and sodium bis (2 ethylhexyl)
sulfosuccinate. In some embodiments the surfactant may be
polysorbate 80, polysorbate 20, sarcosyl, Triton X-100,
.beta.-octyl-gluco-pyranoside, or Brij 35.
[0107] Where the desired protein contains disulfide bonds in the
native conformation it is generally advantageous to include at
least one disulfide shuffling agent pair in the mixture. The
disulfide shuffling agent pair facilitates the breakage of strained
non-native disulfide bonds and the reformation of native-disulfide
bonds. Disulfide shuffling agents can be removed by dialysis.
[0108] In general, the disulfide shuffling agent pair includes a
reducing agent and an oxidizing agent. Exemplary oxidizing agents
oxidized glutathione, cystine, cystamine, molecular oxygen,
iodosobenzoic acid, sulfitolysis and peroxides. Exemplary reducing
agents include glutathione, cysteine, cysteamine, diothiothreitol,
dithioerythritol, tris(2-carboxyethyl)phosphine hydrochloride, or
.beta.-mercaptoethanol.
[0109] Exemplary disulfide shuffling agent pairs include
oxidized/reduced glutathione, cystamine/cysteamine, and
cysteine/cysteine. Additional disulfide shuffling agent pairs are
described by Gilbert H F, (1990) "Molecular and Cellular Aspects of
Thiol Disulfide Exchange." Advances in Enzymology and Related Areas
of Molecular Biology 63:69-172; and Gilbert H F, (1995)
"Thiol/Disulfide Exchange Equilibria and Disulfide Bond Stability,"
Biothiols, Pt A. p 8-28, which are hereby incorporated by reference
in their entirety.
[0110] The selection and concentration of the disulfide shuffling
agent pair will depend upon the characteristics of the desired
protein. Typically the concentration of the disulfide shuffling
agent pair taken together (including both oxidizing and reducing
agent) is from about 0.1 mM to about 100 mM of the equivalent
oxidized thiol, however, the concentration of the disulfide
shuffling agent pair should be adjusted such that the presence of
the pair is not the rate limiting step in disulfide bond
rearrangement.
[0111] Chaotropic agents (also referred to as a "chaotrope") are
compounds, including, without limitation, guanidine, guanidine
hydrochloride (guanidinium hydrochloride, GdmHCl), guanidine
sulfate, urea, sodium thiocyanate, and/or other compounds which
disrupt the noncovalent intermolecular bonding within the protein,
permitting the polypeptide chain to assume a substantially random
conformation.
[0112] Chaotropic agents may be used in concentration of from about
10 mM to about 8 M. The optimal concentration of the chaotropic
agent will depend on the desired protein as well as on the
particular chaotropes selected. The choice of particular chaotropic
agent and determination of optimal concentration can be optimized
by the skilled artisan in view of the teachings provided herein.
Chaotropes can be removed from protein preparations by, for
example, dialysis before using the protein preparation as a
pharmaceutical.
[0113] When used in the present methods, it is often advantageous
to use chaotropic agents in non-denaturing concentrations to
facilitate the dissociation of hydrogen bonds. While a
non-denaturing concentration will vary depending on the desired
protein, the range of non-denaturing concentrations is typically
from about 0.1 to about 4 M. In some embodiments the concentration
is from about 0.1 M to about 2 M.
[0114] In certain embodiments, guanidine hydrochloride or urea are
the chaotropic agents. A single chaotropic agent maybe be used or a
combination of two or more chaotropic agents (e.g., at least two,
at least 3, or 2 or 3 or 4 chaotropic agents).
[0115] Protein solutions can be agitated before and/or during
refolding. Agitation can be performed by methods including, but not
limited to, ultrasound energy (sonication), mechanical stirring,
mechanical shaking, pumping through mixers, or via cascading
solutions.
[0116] The methods described herein can be performed at a range of
temperature values, depending on the particular protein of
interest, in order reduce the subvisible particulates (e.g., in the
0.1 to 10 micron range). For example, the protein can be refolded
(e.g., disaggregated) at various temperatures, including at about
room temperature, about 25.degree. C., about 30.degree. C., about
37.degree. C., about 50.degree. C., about 75.degree. C., about
100.degree. C., or about 125.degree. C. Generally, the temperature
will range from about 0 to about 50.degree. C., about 10 to about
37.degree. C., or about 20 to about 30.degree. C.
[0117] In some embodiments, the temperature can range from about
20.degree. C. to about 100.degree. C. without adversely affecting
the protein of interest, provided that prior to return to room
temperature, the mixture is brought to a temperature at which it
will not freeze.
[0118] Although increased temperatures are often used to cause
aggregation of proteins, when coupled with increased hydrostatic
pressure increased temperatures can enhance refolding recoveries
effected by high pressure treatment, provided that the temperatures
are not so high as to cause irreversible denaturation. Generally,
the increased temperature for refolding should be about 20.degree.
C. lower than the temperatures at which irreversible loss of
activity occurs. Relatively high temperatures (for example, about
60.degree. C. to about 125.degree. C., may be used while the
solution is under pressure, as long as the temperature is reduced
to a suitably low temperature before depressurizing. Such a
suitably low temperature is defined as one below which
thermally-induced denaturation or aggregation occurs at atmospheric
conditions.
[0119] Where the reduction in pressure is performed in a continuous
manner, the rate of pressure reduction can be constant or can be
increased or decreased during the period in which the pressure is
reduced. In some variations, the rate of pressure reduction is from
about 5000 to 2000 bar/1 sec to about 5000 to 2000 bar/4 days (or
about 3 days, about 2 days, about 1 day). In some embodiments, the
pressure reduction may be approximately instantaneous, as in where
pressure is released by simply opening the device in which the
sample is contained and immediately releasing the pressure.
[0120] Where the reduction in pressure is performed in a stepwise
manner, the process comprises dropping the pressure from the
highest pressure used to at least a secondary level that is
intermediate between the highest level and atmospheric pressure.
The goal is to provide an incubation or hold period at or about
this intermediate pressure zone that permits a protein to adopt a
desired conformation.
[0121] In some embodiments, where there are at least two stepwise
pressure reductions there may be a hold period at a constant
pressure between intervening steps. The hold period may be from
about 10 minutes to about 50 hours (or longer, depending on the
nature of the protein of interest). In some embodiments, the hold
period may be from about 2 to about 24 hours, from about 2 to about
18 hours, or from about 1 to about 10 hours.
[0122] In particular embodiments, constant pressure after the
stepwise reduction is from about four-fifths of the pressure
immediately prior to the stepwise pressure reduction to about
one-tenth of prior to the stepwise pressure reduction. For example,
constant pressure is at a pressure of from about four-fifths to
about one-fifth, from about two-thirds to about one-tenth, from
about two-thirds to about one-fifth of the pressure immediately
prior to the stepwise pressure reduction. Where there is more than
one stepwise pressure reduction step, the pressure referred to is
the pressure immediately before the last pressure reduction {e.g.,
where 2000 bar is reduced to 1000 bar is reduced to 500 bar, the
pressure of 500 bar is one-half of the pressure immediately
preceding the previous reduction (1000 bar)).
[0123] Where the pressure is reduced in a stepwise manner, the rate
of pressure reduction (e.g., the period of pressure reduction prior
to and after the hold period) may be in the same range as that rate
of pressure reduction described for continuous reduction (e.g., in
a non-stepwise manner). In essence, stepwise pressure reduction is
the reduction of pressure in a continuous manner to an intermediate
constant pressure, followed by a hold period and then a further
reduction of pressure in a continuous manner. The periods of
continuous pressure reduction prior to and after each hold period
may be the same continuous rate for each period of continuous
pressure reduction or each period may have a different reduction
rate. In some embodiments, there are two periods of continuous
pressure reduction and a hold period.
[0124] In certain embodiments, each continuous pressure reduction
period has the same rate of pressure reduction. In other
embodiments, each period has a different rate of pressure
reduction. In particular embodiments, the hold period is from about
8 to about 24 hours. In some embodiments, the hold period is from
about 12 to about 18 hours.
[0125] Commercially available high pressure devices and reaction
vessels, such as those described in the examples, may be used to
achieve the hydrostatic pressures in accordance with the methods
described herein (see BaroFold Inc., Boulder Co.).
[0126] Multiple-well sample holders may be used and can be
conveniently sealed using self-adhesive plastic covers. The
containers, or the entire multiple-well sample holder, may then be
placed in a pressure vessel, such as those commercially available
from the Flow International Corp. or High Pressure Equipment Co.
The remainder of the interior volume of the high-pressure vessel
may than be filled with water or other pressure transmitting
fluid.
[0127] Mechanically, there are two primary methods of high-pressure
processing: batch and continuous, each of which may be used in
accordance with the invention. Batch processes simply involve
filling a specified chamber, pressurizing the chamber for a period
of time, and repressurizing the batch. In contrast, continuous
processes constantly feed aggregates into a pressure chamber and
soluble, refolded proteins move out of the pressure chamber. In
both set ups, good temperature and pressure control is essential,
as fluctuations in these parameters can cause inconsistencies in
yields. Both temperature and pressure should be measured inside the
pressure chamber and properly controlled.
[0128] There are many methods for handling batch samples depending
upon the specific stability issues of each target protein. Samples
can be loaded directly into a pressure chamber, in which case the
aqueous solution and/or suspension would be used as the pressure
medium.
[0129] Alternately, samples can be loaded into any variety of
sealed, flexible containers, including those described herein. This
allows for greater flexibility in the pressure medium, as well as
the surfaces to which the mixture is exposed. Sample vessels could
conceivably even act to protect the desired protein from chemical
degradation (e.g., oxygen scavenging plastics are available).
[0130] With continuous processing, small volumes under pressure can
be used to refold large volumes the sample mixture. In addition,
using an appropriate filter on the outlet of a continuous process
will selectively release soluble desired protein from the chamber
while retaining both soluble and insoluble aggregates.
[0131] Degassing is the removal of gases dissolved in solutions and
may be advantageous. Gas is much more soluble in liquids at high
pressure as compared to atmospheric pressure and, consequently, any
gas headspace in a sample will be driven into solution upon
pressurization. The consequences are two-fold: the additional
oxygen in solution may chemically degrade the protein product, and
gas exiting solution upon repressurization may cause additional
aggregation. Thus, samples may be prepared with degassed solutions
and all headspace should be filled with liquid prior to
pressurization.
[0132] In certain aspects, the invention provides pharmaceutical
compositions and methods of administration to patients. The
compositions, which may be prepared and/or evaluated by the methods
of the invention, have low immunogenic potential. Immunogenic
potential may be determined by any means known in the art including
antibody titers, relative or absolute amount of antibodies present,
clinical immune reactions such as inflammation and reactions
associated with anaphylaxis (weakness, itching, swelling, hives,
cramps, diarrhea, vomiting, difficulty breathing, tightness in the
chest, lowered blood pressure, loss of consciousness, and shock),
amount of time required for a preparation to provoke detectable
antibodies, amount of time required for a preparation to provoke a
specified antibody titer, and amount of time required for a
preparation to provoke a certain concentration level of antibody.
Antibody titers may be measured by any binding or neutralization
assay known in the art.
[0133] In some embodiments, administration of the pharmaceutical
composition does not result in a loss of immune tolerance to
repeated administrations or endogenous protein. "Tolerance" or
"immune tolerant" as used herein, refers to the absence of an
immune response to a specific antigen (e.g., the therapeutic
protein) in the setting of an otherwise substantially normal immune
system. Tolerance is distinct from generalized immunosuppression,
in which all, or part of, immune responses are diminished.
[0134] The pharmaceutical compositions in accordance with the
invention are formulated so as to have low immunogenic potential.
For example, the amount of protein particulates in a subvisible
range (e.g., about 0.1 to about 50 microns, or about 0.1 to about
10 microns in size) is below a threshold immunogenic dose as
determined, for example, by MFI. As disclosed herein, MFI analysis
showed particulate aggregates in commercial formulations that were
not detectable by SEC or visual inspection, and these solutions
were found to be immunogenic in mice. Particulate doses as low as
1.6 ng/dose broke tolerance in mice and induced immune responses to
monomeric protein. When the preparation was treated with high
hydrostatic pressure the particulates were reduced to a dose level
of 0.02 ng/dose and the immunogenicity was eliminated.
[0135] Thus, the invention provides pharmaceutical compositions and
formulations comprising a therapeutic protein, as well as methods
of treatment with the same. The composition contains subvisible
particulates (e.g., in the range of 0.1 to about 10 microns in
size) at below about 100 ng/dose, below about 50 ng/dose, below
about 10 ng/dose, or below about 2.0 ng/dose. In certain
embodiments, the subvisible particulates are below about 1.5
ng/dose, below about 1.0 ng/dose, below about 0.5 ng/dose, below
about 0.2 ng/dose, below about 0.1 ng/dose, or below about 0.05
ng/dose. The dose may be of any acceptable volume, such as 1 ml in
certain embodiments. Generally, the subvisible particulates are
present below an immunogenic threshold so as not to break
tolerance, or so as to have a low immunogenic potential upon
repeated administration.
[0136] The pharmaceutical composition may be formulated for
administration in a manner that, conventionally, has a tendency to
induce immune reactions to the therapeutic agent, such as
intra-muscular, subcutaneous, or intravenous administration. The
pharmaceutical composition in accordance with the invention has low
immunogenic potential (even for repeated and/or chronic treatment
regimens).
[0137] The therapeutic composition may be indicated for acute or
chronic administration, such as, for example, approximately daily,
weekly, or monthly administration for a period of months or even
years (e.g., from 2 to 20 years). The protein composition may
therefore be administered a plurality of times, including at least
three administrations, at least 5 administrations, at least 10
administrations, at least 20 administrations, at least 50
administrations, at least 100 administrations, or more.
[0138] The therapeutic protein may be indicated for treatment of a
chronic disease, such as diabetes mellitus (e.g., type 1 or type
2), chronic viral infection (e.g., hepatitis A, B, and/or C), or an
autoimmune disorder, such as multiple sclerosis or rheumatoid
arthritis, clotting deficiency, or enzyme deficiency (e.g., PKU).
Other diseases include cancer (e.g., breast cancer, lung cancer,
colon cancer), COPD, and asthma. Exemplary protein compositions for
use in such indications, which may be evaluated for and/or reduced
in their immunogenicity in accordance with the invention, have been
described. By providing therapeutic protein compositions having low
immunogenic potential, the invention allows prolonged therapy
without breaking tolerance and/or without diminishing therapeutic
effect.
EXAMPLES
Example 1
Immunogenicity of Aggregates of Recombinant Human Growth Hormone in
Mouse Models
SUMMARY
[0139] Aggregation of recombinant therapeutic protein products is a
concern due to their potential to induce immune responses. In this
example, the immunogenicity of protein aggregates was examined in
commercial formulations of recombinant human growth hormone
produced by freeze-thawing or agitation, two stresses commonly
encountered during manufacturing, shipping and handling of
therapeutic protein products. In addition, each preparation was
subjected to high-pressure treatment to reduce the size and
concentration of aggregates present in the samples. Aggregates
existing in a commercial formulation, as well as aggregates induced
by freeze-thawing and agitation stresses enhanced immunogenicity in
one or more mouse models. The use of high-pressure treatment to
reduce size and concentrations of aggregates within recombinant
human growth hormone formulations reduced their overall
immunogenicity in agreement with the "immunon" hypothesis.
INTRODUCTION
[0140] Therapeutic proteins are susceptible to aggregation in
response to a wide variety of stresses encountered during their
manufacture, storage and delivery to patients (1). In turn,
aggregates of therapeutic proteins may compromise their safety and
efficacy (2-5). The primary safety concern is that aggregates in
therapeutic protein products may induce immune responses (6,7),
which can have consequences ranging from reduction of product
efficacy to patient fatality (8). In extreme cases,
parenterally-administered aggregates can induce a severe allergic
reaction resulting in anaphylactic shock (9,10). Also, antibodies
formed against aggregated protein molecules have the potential to
cross-react with the native protein as well (5). This
cross-reaction with the native protein may reduce the efficacy of
the therapeutic due to a faster clearance of the protein or
neutralization of the protein. In addition to the neutralization of
the exogenous native therapeutic protein, cases have shown that
antibodies raised against recombinant therapeutic human proteins
can potentially recognize endogenous human proteins (11-13).
[0141] Stresses that frequently provoke protein aggregation such as
agitation (14) or freezing (15) are common in the manufacturing and
shipping of therapeutic proteins. Agitation (and the resulting
exposure of proteins to interfaces such as the air-liquid
interface) can result in aggregation during manufacturing, shipping
and handling of the product (16). Likewise, protein bulk drug
substance is commonly frozen as a storage step in manufacturing
process. Additionally, accidental freezing is a risk, particularly
during refrigerated storage of therapeutic formulations intended
for home use (17). Aggregates produced as a result of different
stresses may exhibit different size distributions and their
component proteins may contain different secondary and tertiary
structures (18), which presumably expose different epitopes and
thus potentially provoke different immune responses (19). Previous
studies reported the immunogenicity of aggregates formed in
interferon .alpha.2 formulations (20,21). In the previous study,
aggregates were generated by oxidation with hydrogen peroxide,
metal-catalyzed oxidization, cross-linking with glutaraldehyde, or
exposure to extreme pH. Conditions that result in aggregation via
oxidation or exposure to extreme pH may be encountered in
industrial processes, but aggregation of therapeutic proteins is
more frequently the result of stresses incurred during
freeze-thawing and agitation. Thus, the current study focuses on
aggregates formed during agitation and freeze-thawing of
recombinant human growth hormone (rhGH) and their potential impacts
on the immunogenicity of the protein. This example also
demonstrates the use of high (.about.2 kbar) hydrostatic pressures
as a method to disaggregate the protein (22,23) with a resultant
decrease in immune response.
[0142] Due to a lack of sophisticated models and a need for greater
understanding of human immune function, pre-clinical predictability
of immunogenicity to recombinant human therapeutic proteins is
problematic (24). Preclinical immunogenicity studies frequently
rely on murine models, in part because mice are relatively
inexpensive and low maintenance and are readily available. Naive
mice inherently develop immune responses to foreign proteins (such
as therapeutic human proteins). However, murine models may
demonstrate enhanced immune responses to more immunogenic samples,
and provide a means by which to assess relative immunogenicity of
various types of aggregates of a given protein (25). Alternatively,
Hermeling et al. (26) recently developed a transgenic mouse model
in which the mice were genetically altered to produce a human
protein in order to eliminate the innate immune response to that
protein, but the relevance of these models to prediction of
responses in humans is also still uncertain.
[0143] In this study we used three murine models to measure the
immunogenic response to protein aggregates produced by agitation or
freeze-thawing stresses in two commercial formulations of rhGH.
Aggregates were characterized for size and conformation of the
component protein molecules. Two murine models used, naive adult
and transgenic, are similar to models used in previous work
(25,26). The third murine model is a neonatally-primed model in
which mice are sensitized to the rhGH in the neonatal stage
(27-29). The neonatally-primed model was chosen to mimic the effect
of low concentration pre-existing antibodies to a therapeutic
protein. It has been reported that antibodies formed during
treatment with a protein therapeutic can be found in the patient in
some cases as long as 59 months after discontinuing treatment with
that therapeutic (30-32). The presence of antibodies to a
therapeutic in a patient after cessation of therapy could pose
unknown risks if the patient were to relapse and need additional
treatment with that therapeutic.
Materials and Methods
[0144] Materials
[0145] The two commercial formulations of rhGH Nordiflex.RTM. (Novo
Nordisk.RTM., Bagsvaerd, Denmark) and Saizen.RTM. (Serono,
Rockland, Mass.), were purchased from the University of Colorado
apothecary, and are hereafter referred to as Product A and Product
B, respectively. Sterile water for injection (SWFI) (Hospira, Inc.,
Lake Forest, Ill.) and 0.9% sodium chloride for injection (Hospira,
Inc., Lake Forest, Ill.) were also purchased form the University of
Colorado apothecary. Histidine and mannitol were purchased from JT
Baker (Phillipsburg, N.J.). Pluronic F-68 was purchased from
Spectrum Chemicals (New Brunswick, N.J.). Phenol was obtained from
Sigma Chemicals (St Louis, Mo.).
[0146] Sample Preparation
[0147] For samples produced from the liquid rhGH formulation
Product A, 15 mg/1.5 ml vials were used for sample preparation. The
rhGH was diluted to a concentration of 1 mg/ml. One of two diluents
was used: (1) a solution of identical composition to the product A
formulation buffer: 1.13 mg/ml histidine, 3 mg/ml pluronic F-68, 3
mg/ml phenol, 19.3 mg/ml mannitol in SWFI at pH 6.5; (2) the
product A formulation without pluronic F-68: 1.13 mg/ml histidine,
3 mg/ml phenol, 19.3 mg/ml mannitol in SWFI at pH 6.5.
[0148] For samples generated from the lyophilized rhGH formulation
Product B, 8.8 mg vials were used for sample preparation. The
lyophilized samples were reconstituted with 3 ml of SWFI resulting
in a formulation containing 2.9 mg/ml rhGH, 20.1 mg/ml sucrose and
0.68 mg/ml o-phosphoric acid at pH between 6.5 and 8.5.
[0149] To induce the formation of aggregates by agitation, 0.6 ml
samples of Product A prepared with diluent 2 or Product B
formulation were pipetted into 2 ml polypropylene tubes, which were
placed horizontally on a Lab-line titer plate shaker and agitated
at approximately 1000 rpm for 72 hours at room temperature. A total
of six 2-ml polypropylene tubes containing 0.6 ml of Product A
prepared with diluent 2 were pooled together to make one large
batch of sample after the 72 hours of agitation. Similarly, the
contents of two 2-ml polypropylene tubes containing 0.6 ml of
Product B formulation were combined after 72 hours of agitation.
The samples processed in this manner are referred to as "agitated
Product A" and "agitated Product B."
[0150] Samples were freeze-thawed (referred to as "FT Product A"
and "FT Product B") by placing 0.75 ml of Product A formulation
(diluent 1) or Product B formulation into each of a total of five 2
ml polypropylenes tubes and two 2 ml polypropylenes tubes,
respectively. The tubes were placed into liquid nitrogen for one
minute to ensure complete freezing of the samples. To thaw the
samples, the tubes were suspended in a water bath at 22.degree. C.
for ten minutes. The freeze-thaw cycle was repeated 20 times and
the appropriate tubes were pooled together to form one batch of FT
Product A and one batch of FT Product B.
[0151] Samples of Product A formulation and Product B formulation
that were not agitated or freeze-thawed were used as controls for
immunogenicity studies. These controls are simply referred to as
"Product A formulation" and "Product B formulation". The samples
were stored at 4.degree. C.
[0152] Disaggregation with High-Hydrostatic Pressure
[0153] The effects of high hydrostatic pressure on rhGH solutions
and solutions containing suspended or soluble rhGH aggregates
(agitated, FT, and formulation samples of Product A or Product B)
were examined by first placing 1.5 ml of pooled Product A samples
and 0.75 ml of pooled Product B samples in Pro-VENT.TM. Caissons
(BaroFold, Inc., Boulder, Colo.). Samples were then loaded into a
Pre-EMT.TM. E150 (BaroFold, Inc., Boulder, Colo.) pressure vessel
at room temperature and pressurized with water. Pressure was
increased at a rate of 0.1 kbar/minute until a pressure of 2 kbar
was achieved. At 2 kbar, the temperature of the high pressure
vessel was increased to 70.degree. C. and the samples incubated for
16 hours. High-pressure in conjunction with high-temperatures
(65.degree. C.) are necessary to overcome intermolecular hydrogen
bonding for proper disaggregation and refolding to occur with human
growth hormone (23). Prior to depressurization (0.1 kbar/min), the
pressure vessel was cooled to room temperature. Samples of agitated
Product A, FT Product A, and Product A formulation treated at high
pressure are referred to as "HP agitated Product A", "HP FT Product
A" and "HP Product A formulation" respectively. Similar notation is
used for high-pressure treated Product B samples.
[0154] Chromatographic Analysis of rhGH
[0155] Monomer and soluble aggregate levels of rhGH were quantified
using size exclusion high performance liquid chromatography
(SE-HPLC). A Superdex.TM. 75 10/300 GL column was used for the
SE-HPLC assay. A Beckman Coulter System Gold HPLC with 126 solvent
module and Waters autosampler were used online with an ultraviolet
detector set at a wavelength of 280 nm. The mobile phase was
phosphate buffered saline (PBS) (2 mM KH2PO4, 10 mM NaH2PO4, 3 mM
KCl, 140 mM NaCl, pH 7.4), and the flow rate was 0.6 ml/min. The
sample injection volume was 50 .mu.l. The samples were kept at
4.degree. C. in the autosampler until injection. Data were
collected over a period of 90 minutes. The chromatograms were then
imported into GRAMS software (Thermo Electron Corp., Waltham,
Mass.) and integrated to determine areas for respective peaks. Peak
area percentages are calculated based on areas obtained through
integrations of SE-HPLC chromatograms. Peak areas percentages were
relative to monomer control peak areas by the following
equation:
Area peak Area monomer control , total .times. 100 Eq . 1
##EQU00001##
Peak area percentages of insoluble aggregates determined through
following mass balance:
Area monomer control , total - Area preparation , total Area
monomer control , total .times. 100 Eq . 2 ##EQU00002##
95% confidence intervals were calculated from triplicate injections
of each sample on the SE-HPLC.
[0156] Analysis of Chemical Degradation Resulting from
High-Pressure Treatment
[0157] Anion exchange chromatography was used to determine
deamidation of rhGH before and after pressurization. The method
used was adapted from a previously published method (33). An
Agilent 1100 HPLC system was equipped with a Tosoh TSK SuperQ-5PW
column and running buffers 10 mM potassium phosphate, 10%
acetonitrile pH 7.4 (A) and 250 mM potassium phosphate, 10%
acetonitrile pH 7.4 (B). The Protein was eluted using a linear
gradient of 0-80% B over 45 minutes. Any remaining protein was
eluting with 100% B wash step followed by a 7 minute equilibration
of 0% B. Absorbance at 280 nm was recorded for 55 minutes.
[0158] Matrix-assisted laser desorption ionization spectroscopy
(MALDI) was performed on a Voyager System (Applied Biosystems,
Foster City, Calif.). The matrix used was
.alpha.-cyano-4-hydroxycinnamic acid.
[0159] SDS-PAGE
[0160] SDS-PAGE was performed on pre-cast tris-glycine
polyacrylamide gels under reducing and nonreducing conditions.
Samples were diluted 2.times. in Invitrogen Novex.RTM. tris-glycine
SDS sample buffer (reducing) or Invitrogen Novex.RTM. tris-glycine
Native buffer (non-reducing) and heated for 5 minutes at 75.degree.
C. A total of 4 .mu.g of protein from each sample in a volume of 10
.mu.l was loaded into the wells of the 1.0 mm 4-20% pre-cast Novex
tris-glycine gel and allowed to run for 1 hour at lab temperature
at 200 volts. The gel was stained with coomassie blue and digital
photos were taken.
[0161] Particle Sizing
[0162] A Beckman Z1.TM. series COULTER COUNTER.RTM. (Fullerton,
Calif.) was used to count particles in solutions containing
insoluble aggregates. The instrument had the ability to detect
particles 1.5 micron and greater. Particles in ranges 1.5-3 micron,
3-6 micron and 6-9 micron were counted five times for each sample.
The counts were averaged and 95% confidence intervals were
determined.
[0163] CD Spectroscopy
[0164] Circular dichroism spectra were obtained for the Product B
samples (Product B formulation, FT Product B, and agitated Product
B) from 190 to 250 nm at 22.degree. C. in a 0.1 cm quartz cuvette
using a Jasco J-810 spectropolarimeter. The spectra were an average
of three measurements with the buffer spectrum subtracted from the
protein spectra. To determine the spectrum for the aggregates in a
sample, the CD spectra were corrected by subtracting the monomer
spectrum multiplied by the amount of monomer present in the sample.
The spectra were then converted to molar ellipticity using a mean
residue molecular weight of 115. The percent of alpha helix, beta
sheet, turns and random coil were determined for each sample using
the SELCON program on the online server DICHROWEB (34). The
structural content for triplicate samples was averaged and 95%
confidence intervals were determined.
[0165] Fluorescence Spectroscopy
[0166] Fluorescence emission spectra for soluble samples were taken
from 300 to 450 nm using a Horiba Jobin Yuon Fluoromax-3
fluorimeter. Triplicate preparations of Product B formulation, FT
Product B and agitated Product B were prepared as described
earlier. Samples were at a concentration of 0.04 mg/ml. Wavelengths
of 260 nm, 280 nm and 295 nm were used for excitation and slits
were set at a 1 nm. An average of 3 scans was taken for each sample
and an appropriate buffer spectrum was subtracted from the protein
spectrum. The emission scans for aggregate preparations were
corrected for monomer signal by subtracting the monomer emission
multiplied by the fraction of monomer in each sample. The center of
spectral mass (CSM) was calculated for each sample using
SigmaPlot.RTM. software (Systat Software Inc., San Jose, Calif.).
The average CSM and 95% confidence intervals were determined for
each preparation (Product B formulation, FT Product B and agitated
Product B).
[0167] 2D-UV Spectroscopy
[0168] UV spectra of soluble rhGH samples were taken from 200 to
500 nm in 1 nm intervals with an integration time of 25 seconds on
a Hewlett Packard 8453 spectrophotometer. Each sample spectrum was
blanked against a buffer spectrum. The aggregated samples were
corrected for monomer content by subtracting the monomer spectrum
multiplied by the fraction of monomer present in that sample.
Second derivatives of spectra were calculated using HP UV-Vis
Chemstation software (Hewlett Packard).
[0169] Fourier Transform Infrared (FTIR) Spectroscopy
[0170] Fourier-transform infrared (FTIR) spectra for native rhGH
and insoluble aggregates of rhGH were acquired using a Bomem.TM. IR
spectrometer (Quebec, Canada) and a dTGS (deuterized triglycine
sulfate) KBr detector. The aggregates were centrifuged at 5,000 rpm
for 5 minutes and the supernatant removed. The aggregates were
resuspended in their appropriate buffer (Product A or Product B) at
a protein concentration of 20 mg/ml. Native rhGH taken directly
from the purchased Product A without dilution at a concentration of
10 mg/ml was used to obtain a FTIR spectrum. A non-diluted sample
of Product A was also pressurized using the same protocol as
described earlier to obtain a FTIR spectrum of high-pressure
treated rhGH. Similarly, a sample of Product B was prepared by
reconstitution to a higher concentration (5 mg/ml) and a sample
pressurized in order to acquire FTIR spectra of the native rhGH in
Product B formulation and high-pressure treated Product B
formulation. A variable path length CaF2 cell was used for the
measurements. The method used to obtain and analyze spectra has
been described previously (35). All mathematical manipulations of
spectra were performed in GRAMS software (Thermo Electron Corp.,
Waltham, Mass.).
[0171] Animals
[0172] Pregnant C57/BL/6 mice crossed with CH3 mice were obtained
from Charles River Laboratories (Raleigh, N.C.). Adult (.gtoreq.6
weeks of age) female B6C3F1 offspring were used for immunogenicity
testing (see below).
[0173] To sensitize the B6C3F1 mice (described above) to rhGH for
use in the neonatally-primed animal model, 10 .mu.l injections
containing 1 .mu.g of rhGH (either Product A formulation or Product
B formulation) were administered intraperitoneally in B6C3F1
neonates for 7 consecutive days with the first injection given
within 24 hours of birth. These mice were caged together and
labeled to separate them from the B6C3F1 naive model animals. Adult
(.gtoreq.6 weeks old) female, primed mice were used for
immunogenicity testing (see below).
[0174] The transgenic mice producing human growth hormone of the
strain B6.SJL-Tg(HBB-GH1)420King/J were purchased from Jackson
laboratories (Bar Harbor, Me.). The mice were acclimated for at
least 7 days before use. Adult (.gtoreq.6 weeks old) female mice
were used for immunogenicity testing (see below).
[0175] Immunogenicity Testing in Animal Models
[0176] Once the naive B6C3F1, neonatally-primed B6C3F1, or
transgenic (B6.SJL-Tg(HBB-GH1)420King/J) mice were of age, blood
was sampled so that each mouse could serve as its own baseline (Day
0). Once a week for five weeks (Days 0, 7, 14, 21, 28) blood was
collected from the retro-orbital venous sinus using 50 .mu.l
Fisherbrand microhematocrit capillary tubes. Mice were sedated with
Isofluorane inhalant gas throughout the blood collection process.
After the blood was collected, each mouse was injected
subcutaneously with 10 .mu.g of human growth hormone (Product B or
Product A) that had been subjected to one of the six conditions
(i.e., agitated, FT, formulation, HP agitated, HP FT or HP
formulation) and diluted in saline for injection (Hospira, Lake
Forest, Ill., lot 49-521-DK) for a total volume of 100 .mu.l. A
total of 8 mice were used in each group. A separate sample of
buffer without protein diluted to 100 .mu.l in saline for injection
(Hospira, Lake Forest, Ill., lot 49-521-DK) was given in each
animal model as a negative control. Additionally, a positive
control of 10 .mu.g of ovalbumin in 100 .mu.l of saline for
injection (Hospira, Lake Forest, Ill., lot 49-521-DK) was given in
the transgenic model. The mice received a total of three injections
on days 0, 7 and 14 after the initial blood collection was
performed. On day 28 the mice were euthanized by exsanguination and
cervical dislocation.
[0177] The collected sera were tested for IgG specific antibody
response using an enzyme-linked immunoassay (ELISA). The wells of
Immulon 4 High Binding Affinity plates (ISC Bioexpress, Kaysville,
Utah) were incubated with 200 .mu.l of a diluted monomeric rhGH
solution (16 .mu.g/ml) prepared from the Product A or Product B
formulations at lab temperature overnight with gentle agitation.
The wells were then drained and washed three times with PBS. After
the final wash the wells were tapped dry on a paper towel.
[0178] The wells were then blocked with 200 .mu.l of 1% bovine
serum albumin (BSA) in PBS for 1 hour. After application of the
blocking solution the wells were washed three times with a solution
of PBS. Wells in rows B-H were then loaded with 100 .mu.l of
dilution buffer (40 mM HEPES, 10 mM disodium EDTA, 150 mM sodium
chloride with 1% BSA and 0.1% triton.times.100). The sera were then
diluted 1:20 into the dilution buffer and added to the wells in row
A. Each plate had two standard curves of known concentration of
mouse monoclonal antibody [GH-2] to human growth hormone (Abcam
ab9822, Cambridge, Mass.). Using a multichannel pipet, 100 .mu.l of
the diluted sera from row A were transferred to the wells in row B.
The solution in row B was then mixed by drawing up and expelling
100 .mu.l (5 times) into the wells before transferring 100 .mu.l to
wells in row C. The 2.times. dilutions were continued through row
G. The plates were then sealed and allowed to incubate at lab
temperature for 30 minutes. Then, the wells were washed 3 times
with a solution of 40 mM HEPES, 10 mM disodium EDTA, 150 mM sodium
chloride and 0.1% Triton.times.100 and tapped dry on a paper towel.
The wells were incubated with 100 .mu.l of a horse radish
peroxidase conjugated goat anti-mouse IgG (Chemicon, AP308A,
Temecula, Calif.) diluted 1:8000 in dilution buffer. After 1 hour,
the wells were washed three times with PBS and tapped dry on a
paper towel, followed by the addition of 100 .mu.l of 3,3',5,5'
tetramethylbenzidine to each well. After 20 minutes, 50 .mu.l of
0.5 M sulfuric acid were added to the wells to stop the reaction.
The absorbance was recorded with a Molecular Devices (Sunnyvale,
Calif.) "V max" kinetic plate reader at a wavelength of 450 nm and
a reference wavelength of 595 nm. The ELISA response was reported
in units of ng/ml and was calculated from the average absorbance
response on a standard curve multiplied by its dilution factor. The
standard curve (r2 value of 0.99) was generated from the standards
on each plate using a four-parameter fit in Softmax (Sunnyvale,
Calif.) with an antibody concentration range of 1250 ng/ml to 10
ng/ml.
[0179] ELISA assays were also performed using Immulon 4 plates
coated high-pressure treated rhGH (16 .mu.g/ml) to determine if any
differences in immune responses were observed between pressurized
and non-pressurized material. Serum was analyzed using the same
protocol as described above.
[0180] The data were modeled as a general factorial design with 1
response and levels appropriate to the number of groups in each
study. Each sample group had eight replicates. The software program
Stat-Ease 7.2.1 (Minneapolis, Minn.) was used to conduct a linear
analysis of variance (ANOVA). The probability of a [p] between
means of groups was compared with a 90% confidence interval. When
comparing means, probabilities of [p]<0.1 were significant based
on the 90% confidence interval chosen.
Results
[0181] Stressing of rhGH Samples
[0182] The responses of the Product A and Product B formulations to
the various stresses (agitation, freeze-thawing) were different.
The agitated Product A samples were cloudy by the end of the 72
hours of agitation whereas the agitated Product B samples were
still clear. Similarly, the FT Product A samples began to become
cloudy around the 12-15th freeze-thaw cycle; however, the FT
Product B never showed signs of cloudiness even after the 20th
freeze-thaw cycle. The contrast in aggregates produced in the two
samples is believed to be caused by the presence of phenol in
formulation A. Phenolic compounds have been shown previously to
induce the formation of large insoluble aggregates of rhGH.36
[0183] Characterization of Aggregates within rhGH Samples
[0184] SE-HPLC was used to determine the aggregation state of each
sample type. In Product B formulation, no soluble or insoluble
aggregates could be detected (FIG. 1). After application of
freeze-thawing or agitation stresses, levels of soluble aggregates
increased to 31% and 69% respectively. High pressure treatment of
FT Product B and agitated Product B resulted in a substantial
increase in monomer levels and soluble dimer aggregate compositions
of 4 and 3%, respectively (FIG. 2). The product B formulation
sample treated with high pressure had a soluble dimer aggregate
content of 1%.
[0185] The SE-HPLC chromatograms for the Product A formulation, FT
Product A and agitated Product A samples used in the naive adult
and primed naive animal models are shown in FIG. 1. Product A
formulation produced insoluble aggregates after freeze-thawing or
agitation that could not be injected onto the SE-H PLC, but could
be quantified by mass balance based on the starting protein
concentration and that represented in the chromatograms. The
Product A formulation contained 2% soluble aggregate, even before
being subjected to agitation or freeze-thawing stresses (FIG. 1).
From the enlarged portion of the chromatogram it appears that these
aggregates are composed mostly of dimer with relatively smaller
amounts of higher molecular weight oligomers. The agitated Product
A sample contained 12% soluble aggregate and 42% insoluble
aggregate. FT Product A had just 5% soluble aggregate and 72%
insoluble aggregate. All three Product A preparations (formulation,
agitation and FT) exhibited soluble aggregates in the void volume
of the Superdex 75 10/300 column (.about.700 seconds) which
indicates aggregates with molecular weights larger than 100,000.
After high pressure treatment there was a significant increase in
the level of monomer for both the agitated Product A and FT Product
A samples, with post-pressure treatment soluble aggregate contents
of 7 and 4% respectively (FIG. 2). No insoluble aggregates were
detectable in the high pressure treated samples. The high pressure
treated Product A formulation (HP formulation) had soluble dimer
aggregate content of 2%.
[0186] Samples used in the transgenic animal model were prepared
separately from those in the naive and primed models and resulted
similar aggregate types and levels for the different treatments
(FIG. 3).
[0187] All preparations were analyzed by SDS-PAGE to test for the
presence of covalent aggregates (data not shown). No preparation
for any formulation contained detectable levels of covalently
linked aggregates.
[0188] The secondary structures of protein molecules in the
insoluble aggregates produced in Product A formulation were
determined using infrared spectroscopy. The protein in aggregates
produced through agitation and freeze-thawing retain the native
alpha-helical secondary structure as shown by the strong signal at
1654 cm.sup.-1 in FIG. 4. Even though the stresses produce large
insoluble aggregates, the overall secondary structure of the
component rhGH molecules was minimally perturbed. The secondary
structure of Product A and Product B before and after high-pressure
treatment was also determined using infrared spectroscopy. No
differences were observed in the secondary structures of Product A
formulation and its high-pressure treated counterpart even though
deamidation occurs during high-pressure treatment (Data not shown).
Similarly, pressure treated Product B formulation retained native
alpha-helical structure (Data not shown).
[0189] The size and number of particles produced in the aggregated
preparations of Product A were determined through the use of a
Beckman Coulter Counter. The lower limit of detection was 1.5
micron. The FT Product A sample had 97% of its particles in the
1.5-3 micron range and 3% in the 3-6 micron range. There were no
detectable particles in the 6-9 micron range for the FT Product A
sample. The agitated Product A sample had 85% 1.5-3 micron
particles, 14% 3-6 micron particles and 1% 6-9 micron particles.
Neither the FT Product A sample nor the agitated Product A sample
had particles detectable larger than 9 micron.
[0190] CD spectra were obtained for the soluble Product B
preparations that were made in triplicate as a method to analyze
the secondary structure of the aggregates (FIG. 5). Light
scattering from insoluble aggregates prevented the use of CD
spectroscopy for analysis of aggregated Product A samples. The
agitated Product B preparation retains more of the alpha helical
content than the FT Product B preparation, with compositions of 59%
and 54% respectively. The FT Product B has more .beta.-sheet
content than the agitated Product B and Product B formulation
samples. Both the FT Product B and agitated Product B samples have
at least a 2% increase in random coil structures compared to the
Product B formulation preparation.
[0191] UV scans of the soluble aggregate preparations were used in
the characterization of tertiary structure of protein in the
stressed Product B samples. Product B samples were made in
triplicate and 95% confidence interval were calculated for the mean
measurements. Product A samples could not be analyzed with UV due
to light scattering caused by the insoluble aggregates. The peak
positions in second derivative UV scans from 270-300 nm can be used
to describe the tertiary structure of proteins by identifying the
microenvironments around tyrosine (Tyr) and tryptophan (Trp)
residues (37,38). The spectra for the protein in both the agitated
Product B and FT Product B samples had red shifts in the tyrosine
peaks with the most predominant shifts observed in spectrum for the
FT preparation. Human growth hormone molecules contain eight
tyrosine residues. It is clear from the red-shifts observed in the
two aggregated samples that one or more tyrosines are more exposed
to more apolar environments indicating a change in tertiary
structure. The single tryptophan located in an .alpha.-helical
region of the protein has little change in its microenvironment as
evidenced from the constant position of the peak at 293 nm.
[0192] Fluorescence spectroscopy also can be used as a method to
monitor protein tertiary structure. Different excitation
wavelengths can be used to monitor the change in microenvironments
of tyrosine and tryptophan residues. Due to light scattering from
insoluble aggregates, Product A samples could not be analyzed with
fluorescence. Emission scans of the soluble Product B formulation,
FT Product B and agitated Product B were recorded at excitations
wavelengths of 260, 280 and 295 nm. The wavelength at which the
highest fluorescence intensity was observed (.lamda.max) was
determined for each emission scan. Each preparation was made in
triplicate and 95% confidence intervals were calculated for the
mean. Slight red-shifts were observed in .lamda.max for the
emission scans of agitated Product B and FT Product B samples at an
excitation wavelength of 260 nm indicating slight alterations in
the tertiary structure around the tryptophan residue. However, the
Amax at an excitation wavelength of 295 nm is not statistically
different between the three sample types. Therefore, in agreement
with second derivative UV spectroscopy the Amax at an excitation of
295 nm indicates that the aggregates formed in both FT and agitated
Product B formulations have minimal change in the .alpha.-helix
containing the molecule's single tryptophan.
[0193] Analysis of Chemical Degradation Resulting From
High-Pressure Treatment
[0194] Deamidation levels were measured before and after
pressurization by anion exchange chromatography. The percentage of
deamidated rhGH was found to increase upon pressurization. For
Product A the amount of deamidated protein increased from 6 to 60%
subsequent to high-pressure treatment. Product B experienced an
increase from 2 to 92% after being subjected to high pressure.
MALDI experiments detected changes in molecular weight consistent
with deamidation in sample subjected to high-pressure treatment. No
evidence of other chemical degradation (e.g. oxidation or
fragmentation) was observed.
[0195] Immunogenicity
[0196] In naive adult and neonatally-primed mice, maximum levels of
antibodies were observed in serum samples collected at week 4 (FIG.
6). However, the three animal models generated different levels of
antibody production throughout the course of the study. The
neonatally-primed mice exhibited anti-hGH IgG levels of mg/ml
whereas the naive adult mice resulted in .mu.g/ml antibody
production. The transgenic mice had no detectable anti-hGH IgG
antibodies present in any of the treatment groups.
[0197] Immune responses were measured using ELISA plates coated
with native rhGH or high-pressure treated rhGH. No statistically
significant differences of antibody titers were observed between
the two types of plates (Data not shown). Therefore, the results
that follow are given in titers against native rhGH that was not
subjected to high-pressure treatment.
[0198] All preparations of Product A and Product B induced an
immune response in the neonatally-primed mice. The increased level
of anti-hGH IgG, as well as the presence of antibodies at week 1,
prior to first inoculation (FIG. 6) in the primed model suggests
that sensitization was achieved. The negative control of buffers
alone did not induce a response. Even though all animals produced
antibodies to human growth hormone, some treatment-based
differences were observed. For Product A samples, neither of the
stressed samples (agitated Product A, FT Product A) produced a
significantly higher immune response than the Product A
formulation. While the average immune response of FT Product A was
higher than Product A formulation, it can not be stated as
statistically different (p=0.8577). Product A formulation actually
induced a higher immune response than the agitated Product A
(p<0.0001). The high-pressure treated counterparts of these
samples reduced the immunogenicity of the Product A formulation
(p<0.0001) and FT Product A (p<0.0001); however, the agitated
Product A treated with high pressure did not statistically reduce
the immune response (p=0.2348) (FIG. 7). It can also be stated that
the high-pressure treated samples of Product A (Product A
formulation, agitated Product A, FT Product A) produced immune
responses that are not significantly different. The immune
responses to stressed Product B preparations (FT, agitated) were
not significantly different than that of the Product B formulation.
Additionally, when the Product B preparations were treated with
high-pressure the immune response did not statistically change
(FIG. 7). The immune responses produced from HP Product B
formulation, agitated Product B, and FT Product B were not found to
be significantly different.
[0199] The naive adult mice produced similar results to the
neonatally-primed mice. Again, the negative control of formulation
buffer alone for both the Product A and Product B failed to induce
an immune response. For Product A preparations, the FT Product A
induced a significantly higher immune response than that of the
Product A formulation (p<0.0001). The agitated Product A
produced a lower average immune response than the Product A
formulation, however the responses are not significantly different
(p=0.1979).
[0200] The immune responses for Product A formulation and FT
Product A samples were significantly higher than the immune
responses generated by their high-pressure treated counterparts (HP
Product A formulation, HP FT Product A) (FIG. 8). The immune
responses for agitated Product A and HP agitated Product A are not
significantly different. Again, similar to the neonatally-primed
mice the statistically similar immune responses were generated from
all three high-pressure treated Product A preparations and are not
significantly different. For the Product B preparations, neither
the agitated Product B nor the FT Product B produced statistically
different immune responses than that of the Product B formulation
(p=0.1545 and 0.3870 respectively). The only high-pressure treated
Product B preparation to have a lower immune response than its
counterpart was the HP agitated Product B (p=0.0925). The immune
responses generated from all high pressure treated Product B
samples were not significantly different.
[0201] A group of 8 mice were injected with ovalbumin as a positive
control and successfully induced antibodies. The positive control
produced anti-ovalbumin IgG in the .mu.g/ml levels consistent with
the responses in the naive adult mice indicating that the
transgenic animals had normal immune function. However, no immune
response to any of the rhGH samples was detected in any of the
transgenic animals.
Discussion
[0202] Parenteral administration of aggregates of a therapeutic
protein can induce immune responses to the monomeric protein.
However, little is known about the characteristics of aggregates
that are capable of inducing immunogenicity and the mechanism by
which they provoke the response (5). B cells can be stimulated to
produce antibodies in T-cell independent mechanism that requires an
antigen with a repetitive structure (39). Dintzis et al. determined
that in order for polymeric antigens to activate B-cells
independent of T-cells, the antigens are required to have a minimum
number of antigenic receptors (10-20) with a characteristic
spacing, which is referred to as an "immunon" (40). Dintzis et al.
observed that immunons greater than 100 kDa in molecular weight
with an epitope spacing of approximately 100 angstroms were
successful in inducing B-cell activation (40). The antigenicity of
highly ordered repeating epitopes has been confirmed in subsequent
studies using virus-like particles (VLPs) with epitope spacing of
50-100 angstroms to activate B cells independent of T cells
(41,42). Based on these observations, aggregates of therapeutic
proteins with similar sizes, repeating epitope content and
retention of near-native protein structure also might serve as
antigens to induce an immune response (5,43). Thus, we anticipate
that parenteral administration of aggregates that are both
sufficiently large (>100 kDa) and that retain significant native
structure may result in immunogenicity.
[0203] Immune responses may also be co-stimulated by administering
adjuvants along with protein antigens (44). Adjuvants attract
phagocytic cells such as dendritic cells and enhance their
activation (45). Activated dendritic cells can in turn activate T
cells into cytotoxic T cell or helper T cells (Th1 and Th2) (46).
Adjuvants such as alum increase Th2 response and result in
increased B cell activation and consequent antibody production
(47). Likewise, large particulate aggregates contaminating a
therapeutic product can influence immune responses by enhancing a
Th2 responses (25). We speculate that very large insoluble protein
aggregates could act in a similar, "self-adjuvanting" fashion. Not
only could large insoluble aggregates attract dendritic cells, but
there is potential for dendritic cells to take up the aggregate
through phagocytosis and cleave the molecule into peptide fragments
to be presented on MHCs. Because the aggregate is formed from
multiple protein molecules, the peptide fragments presented on MHCs
could be consistent with those found on the native monomeric
protein, which in turn would enhance the recognition of monomeric
protein molecules as antigens.
[0204] In our analysis, a commercial formulation of Product A was
found to contain 2% soluble aggregates by SE-HPLC. Previous
publications have also found Novo Nordisk.RTM. rhGH products to
contain between 1-5% aggregates (48) and degradation byproducts
related to both oxidation and deamidation (49). The Product A
formulation contained higher-order oligomers that eluted near the
void volume of the sizing column, indicating that the aggregates
had molecular weights .gtoreq.100 kDa. Previous studies have also
shown that aggregates of human growth hormone that eluted in void
volume fractions of a Sephadex.RTM. gel filtration column were
responsible for inducing antibodies in human patients (6). It has
been reported that the human growth hormone molecule has 10
epitopes on its outer surface recognizable by monoclonal antibodies
(50). Taking into consideration the number of epitopes on the hGH
molecule and the sizing results from SE-HPLC, the aggregates found
in the Product A formulation sample meet the immunon criteria for
B-cell activation. Consistent with these observations Product A
induced a substantial immune response in the naive adult and
neonatally-primed mice.
[0205] FT Product A induced the highest immune responses observed
in this study when administered to naive and primed mice. This
sample had large insoluble aggregates containing native-like
secondary structure, as well as presence of smaller soluble
aggregates eluting in the dimer and oligomer (MW>100 kDa)
volumes of SE-HPLC. We speculate that the large insoluble
aggregates could act as adjuvants as well as antigens, thus
resulting in the observed high immune response. In the naive adult
mice, FT Product A exhibited an enhanced immune response compared
to the Product A formulation (p<0.001). In the neonatally-primed
mice, elevated immune responses were seen against both the FT
Product A and the Product A formulations, but the differences in
responses between the two were not statistically significant
(p=0.86). We speculate that because the secondary response shown by
neonatally-primed mice does not require adjuvant (51), the presence
of large aggregates in the FT Product A formulation did not enhance
the immune response in these mice.
[0206] After Product A formulation was treated with high pressure,
only dimer-size soluble aggregates remained, reducing the molecular
weight of aggregates below the .apprxeq.100 kDa size required for
immunon activity. This sample also resulted in a significantly
lower immune response than the untreated counterpart (p<0.0001)
in neonatally-primed and naive mice. Reducing the aggregates with
high-pressure treatment and concomitantly the immune response
documents that aggregates were responsible for the immune response
to Product A formulation.
[0207] Similarly, when FT Product A was treated with high pressure
the molecular weight and concentrations of aggregates were reduced.
The total aggregates content was reduced from 77% to 4% and the
remaining aggregation was of dimer-size. No insoluble aggregation
was present after high-pressure treatment. Reduction of aggregate
molecular weight below 100 kDa and elimination of insoluble
aggregates would eliminate the antigenic and adjuvant-like
properties that exist in the FT Product A sample. Consistent with
this finding is the significantly lower immune response elicited by
the HP FT Product A when compared to the immune response of the FT
Product A sample (p<0.0001) in both the naive adult and
neonatally-primed mice.
[0208] The pressure treatment resulted in increased deamidation
levels for both formulations. Deamidation levels in Product A
increased from 6% to 60%, whereas deamidation levels in Product B
increased from 2% to 92% after pressure treatment. The changes in
deamidation levels did not correlate with immune response, and
equivalent immune responses were detected using ELISA plates coated
with either as-received material or pressure treated protein. Mass
spectrometry did not detect chemical degradation beyond
deamidation. Furthermore, infrared spectroscopy showed that
secondary structure of the protein was unchanged by high-pressure
treatment. Thus, it appears unlikely that pressure induced
conformational or chemical changes in the bulk soluble protein
fraction are responsible for the observed changes in immunogenicity
before and after pressure treatment.
[0209] Agitation and freeze-thawing of Product B did not result in
a statistically significant increase in the immune response in
either neonatally-primed or naive mice even though these treatments
induced formation of soluble aggregates. This may be due to the
lack of an adjuvanting effect because insoluble aggregates were not
present. High-pressure treatment of the agitated Product B
significantly reduced the amount and size of aggregates as well as
the immune response in naive mice. Again, this finding is
consistent with the "immunon" hypothesis as stated previously by
eliminating the larger soluble antigenic aggregates.
[0210] Some of the samples, such as FT Product B and agitated
Product B contained aggregates (31 and 53%, respectively) but did
not result in statistically increased immune responses compared to
unstressed formulations. The soluble aggregates of the FT Product B
sample, which eluted as dimers, trimers and oligomers (>100 kDa)
showed a partial loss of native structure in the component protein
molecules. Thus, an explanation for the lower immune response to FT
Product B could be the loss of native epitopes as well as a lack of
insoluble aggregates that with adjuvanting capacity. Likewise, the
overall lower immune response of agitated Product A in both the
naive adult and neonatally-primed mice could indicate that the
soluble oligomer aggregates present in this preparation did not
expose the same native epitopes as those found in the Product A
formulation and FT Product A samples. However, due to the low
concentration of these aggregates in the samples, the secondary
structure of protein within the aggregates could not be
characterized.
[0211] In the naive mice, antibody levels were generally in the
.mu.g/ml concentration range. In the neonatally-primed mice,
however, antibodies were produced at mg/ml concentrations which is
consistent with levels of antibodies produced in a secondary
response. This is expected since the mice used in the
neonatally-primed model had been given injections of the monomeric
protein previously. The neonatally-primed mice also had a shorter
lag phase in antibody production which is characteristic of a
secondary response. It is important to note that previous
publications describe injections at a neonatal stage as a means to
induce tolerance to a foreign protein rather than sensitization
(52-56). However, our protocol successfully primed the mice at a
neonatal stage allowing an enhanced immune response when given
booster inoculations at an adult stage. Recent studies suggest that
tolerance which had previously been defined as the reduced or lack
of lymph node proliferation of T-cells (57,58) does not equate to
immune tolerance. This is because neonatal injections induce memory
T-cells to the antigen which accumulate in the spleen rather than
in lymph nodes (58,59).
[0212] Interestingly, the transgenic mice failed to produce an IgG
immune response to any of the recombinant human growth hormone
samples. In contrast, previous studies using interferon .alpha.2b
transgenic mice showed that tolerance could be broken in response
to administration of aggregated protein (21). Important differences
between the current study and the previous studies may be related
to the natural function and abundance of the respective proteins
(5), as well as the mouse strains (60) used to generate the
transgenic mice. The current study used the strain
B6.SJL-Tg(HBB-GH1)420King/J, which results from the cross breeding
of C57BL/6J with SJL strains, whereas the interferon .alpha.2b
study was based on the strain FVB/N (61). Both the level of
transgenic protein produced and the robustness of B-cell repertoire
are strain-dependent. The strain used in our study produced human
growth hormone ca. 195 ng/ml under control of a sheep hemoglobin
betac promoter (62). The levels of human interferon .alpha.2b
produced in the transgenic line used in previous studies were not
published. In addition to the differences in transgenic lines
created, the protein that is being produced may have an impact on
the resulting immune responses. Both B-cells and T-cells have
receptors generated by random chromosomal rearrangements that are
specific to antigens that may or may not be encountered through an
individual's lifetime (44). In some cases chromosomal
rearrangements result in receptors on B-cells and T-cells that
identify self molecules. In early development these lymphocytes are
deleted from the repertoire in a process referred to as clonal
selection (46). It is easier to break tolerance to endogenous
proteins found at low levels due to possible incomplete clonal
deletion of lymphocytes reactive to these proteins during clonal
selection (63). Interferon .alpha. is generally expressed at low,
nearly undetectable levels (<3 IU/ml) in healthy humans (64,65);
whereas growth hormone is a more abundant protein, especially
earlier in life with a cyclic production varying from 10-40 .mu.g/L
and decreasing to 4 .mu.g/L as adults (66,67). In addition to
strain-related differences between the current study and the
earlier interferon .alpha.2b transgenic study, the different
methods used to form aggregates may provide an explanation for the
lack of immune response to aggregated rhGH seen in our transgenic
mice. The previous study relied on relatively harsh treatments such
as metal catalyzed forced oxidation, cross-linking with
glutaraldehyde, extreme thermal treatments, and extreme pH to
create an aggregate population. These treatments may have created
additional and/or more potent epitopes in the aggregates than those
of the relatively gentler freeze-thawing and agitation treatments
applied in this study.
[0213] It is important to note that in this study we used
accelerated aggregation techniques to produce aggregates in samples
of commercial therapeutic proteins. A therapeutic protein will most
likely not experience 20 freeze-thaw cycles or agitation for 72
hours; however, these techniques were necessary to produce enough
aggregation to elicit immune response differences in a short time
frame. The objective was simply to determine if differences in
immunogenicity could be observed from aggregates produced through
different stresses. We also chose to only detect immune responses
against native monomeric protein. There may have been antibodies
not detected that were specific to aggregates of monomeric protein.
However, we were most concerned with aggregates that would induce
antibodies that have sufficient crossover recognition of the native
monomeric protein due to their clinical relevance. Antibodies that
only detect aggregates of therapeutic protein may or may not affect
the efficacy of the therapeutic product. Antibodies that recognize
native monomeric protein are much more of a concern for the safety
and efficacy of a therapeutic product. Aggregates with significant
native-like structure would more likely produce antibodies that
could be cross-reactive with native monomeric protein. Our
immunogenicity data and spectroscopic data are consistent with the
premise that native-like aggregates could induce cross-reactive
antibodies to native monomeric protein.
CONCLUSIONS
[0214] This study has shown that the immunogenicity of protein
aggregates may depend on their size and the manner and solution
conditions by which they are produced. Aggregates found in existing
commercial formulations were immunogenic in the naive adult and
neonatally-primed mice, as were aggregates generated by
freeze-thawing- or agitation-induced stresses applied to the
formulations, two types of stresses that are routinely encountered
during production, handling, and storage of protein formulations.
Further, the stock formulation did not contain aggregates (by SEC),
but was immunogenic due to subvisible particulates. Use of high
pressure to reduce aggregate levels reduced immune response
consistent with the immunon hypothesis.
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Example 2
Aggregates of Recombinant Murine Growth Hormone Break Tolerance: A
Model for Adverse Immunogenicity of Therapeutic Proteins
[0283] Recombinant therapeutic proteins now comprise front-line
clinical treatments for many diseases and disorders. Dozens of
therapeutic protein products are approved and marketed and hundreds
more are in clinical trials (1). In spite of the clinical efficacy
of these products, for many of them a drawback is the risk of
adverse immune response (2). Patients that produce neutralizing
antibodies as a result of the immune response may experience
reduced efficacy of treatment and risk serious complications (3).
Furthermore, in some cases the immune response also can neutralize
the endogenous counterpart of the therapeutic protein, causing
permanent harm or death (4,5). In addition, products of great
potential benefit to patients may fail to be approved because of
immunogenicity problems arising during clinical trials.
[0284] Although the mechanisms by which therapeutic proteins may
induce adverse immune responses are not well understood, protein
aggregates may play a role (3). Unfortunately, therapeutic proteins
aggregate throughout their product life cycle, and proteins are
thought to be thermodynamically unstable with respect to
aggregation (6). In addition, aggregation may be accelerated by
stresses such as freeze-thawing or exposure to the air-water
interfaces and other surfaces which are commonly encountered during
manufacturing, shipping, storage and delivery to patients (7,8).
The characteristics of protein aggregates can vary depending on
many factors, including the nature of the initiating stress,
solution conditions, protein physicochemical properties, etc (9).
For example, exposure to air-water interfaces can sometimes cause
the formation of large, soluble oligomers, whereas in other cases
insoluble precipitates and/or subvisible particles are formed.
Also, the conformation of the protein molecules within aggregates
can vary from native-like to greatly perturbed structure (10).
Immunogenicity of protein aggregates may depend on the size of
aggregates, the structure of protein molecules that make up
aggregates and the route of administration (11-13). It has been
hypothesized that aggregates may be of sufficient size and also may
exhibit repetitive epitopes necessary to induce T cell-independent
B cell activation, and the highly ordered, repetitive nature of
some protein aggregates may cause the immune system to respond to
them as if they were pathogen-associated molecular patterns
(PAMPs), thereby inducing innate and adaptive immune responses (3).
Furthermore, particulate protein aggregates and protein molecules
adsorbed on the surface of foreign microparticles could behave as
self-adjuvants (11) enhancing uptake by macrophages and inducing
inflammatory co-stimulatory cytokines, leading to strong T-cell
dependent immune responses. We hypothesize that aggregates formed
as a result of different types of stresses may provoke different
levels and types of immune responses.
[0285] Proteins also may form aggregates by adsorption to micro-
and nanoscopic foreign materials that are commonly found in
therapeutic protein products (14). Patients may be exposed to
foreign particles that are introduced into therapeutic products
during the filling of product containers such as vials or syringes
and/or shed from the product containers themselves (15). For
example, stainless steel particles are eroded from some types of
high-speed filling pumps, and product container/closures can shed
particles of glass, silicone oil, rubber and/or tungsten particles
into formulations (16). In vaccine formulations, protein molecules
typically are adsorbed onto foreign microparticles of materials
such as aluminum salt adjuvants in order to stimulate desirable
immune responses against foreign proteins (17). Our second
hypothesis is that breaking of immune tolerance may result from
administration of therapeutic protein molecules adsorbed onto
commonly-found foreign microparticles.
[0286] Previous studies have used murine models to investigate the
immunogenicity of aggregates of recombinant human therapeutic
products (11, 13, 18-20). In these studies, parenteral
administration of aggregates was often associated with elevated
immune responses compared to that noted with the monomeric protein.
Because human proteins are non-self to mice, some studies have used
transgenic mice to examine breaking of tolerance, whereas others
have used naive mice in which there are innate immune responses to
foreign protein and monitored the level of the response. In this
study, we used recombinant murine growth hormone (21) (mGH) to test
the ability of aggregates of an endogenous protein to break immune
tolerance in mice. We investigated the immunogenicity of protein
aggregates that were formed as a result of two
pharmaceutically-relevant stresses, agitation and freeze-thawing,
as well as the immune response to mGH adsorbed onto microparticles
of glass or alum. To examine the effects of reduced aggregate
content, we applied high hydrostatic pressure to disaggregate
aggregates (22) found in untreated control mGH ("stock"), agitated,
and freeze-thawed samples. Aggregates were quantified and
characterized by size exclusion high performance liquid
chromatography (SE-HPLC), micro-flow imaging particle counting and
optical spectroscopy methods.
[0287] To gain insight into the mechanism(s) through which the
aggregates provoke immune responses (e.g., T-cell dependent or
T-cell independent pathways), we measured the levels of IgG
isotypes produced in the mice.
Materials and Methods
[0288] Materials
[0289] The mGH was produced and purified as described earlier (21).
Alum (Alhydrogel.TM. aluminum hydroxide made by Brenntag Biosector)
was purchased from E.M. Sergeant Pulp &Chemical Co., Inc.
(Clifton, N.J.). Glass microparticles were produced from ball
milling of syringe barrels (Becton Dickinson, Franklin Lakes, N.J.)
as described earlier (14). Saline for injection (Hospira, Inc.,
Lake Forest, Ill.) was purchased from the University of Colorado
apothecary. Goat anti-mouse IgG1, goat anti-mouse IgG2a, goat
anti-mouse IgG2b, goat anti-mouse IgG2c, goat anti-mouse IgG3, HRP
conjugated rabbit anti-goat IgG, and mouse anti-ovalbumin were all
purchased from Abcam (Cambridge, Mass.). 3,3',5,5'
tetramethylbenzidine was purchased from KPL (Gaithersburg, Md.).
All other reagents were from Fisher Scientific (Pittsburgh,
Pa.).
[0290] Sample Preparation
[0291] The stock sample was stored at 4.degree. C. and was used to
prepare all samples of mGH. Aggregates of mGH were formed by
freeze-thawing and agitation stresses. The freeze-thaw sample was
subjected to 20 freeze-thaw cycles of freezing in liquid nitrogen
for 1 minute followed by 10 minute thaw in a 25.degree. C. water
bath. The agitated sample was prepared by securing the sample
horizontally to a Lab-line titer plate shaker and agitating at
approximately 1000 rpm for 4 hours at room temperature. mGH was
adsorbed to alum and glass particles by adding appropriate masses
of particles to protein solution and subjecting them to
end-over-end rotation at 8 rpm for 30 minutes. The mGH was adsorbed
to alum at a 1:1 mass ratio. mGH was adsorbed to the glass at a
mass ratio of 1:76. All samples were kept at 4.degree. C.
[0292] High Pressure Treatment of Samples
[0293] The stock, agitated and freeze-thaw samples were each
subjected to high hydrostatic pressure to dissociate non-covalent
aggregates and refold the protein to the native monomeric state.
Samples were placed in heat-sealed BD syringes and pressurized to
200 MPa over 20 minutes in a Pre-EMT150.TM. pressure vessel
(BaroFold Inc., Boulder, Colo., USA) and held at this pressure for
4 hours at room temperature. The samples were then de-pressurized
over 20 minutes to atmospheric pressure. The samples were then
stored at 4.degree. C. The high pressure treated samples are
referred to as "HP stock", "HP agitated" and "HP freeze-thaw".
[0294] SE-HPLC
[0295] Experiments were conducted using a Superdex.TM. 75 10/300 GL
column on an Agilent 1100 series HPLC system (Agilent Technologies,
Inc., Santa Clara, Calif., USA). Isocratic chromatography was
performed at room temperature with a flow rate of 0.8 ml/min with
100 mM Acetate 100 mM NaCl pH 4.75 as the mobile phase. UV signal
at 280 nm was monitored using the Agilent UV diode array detector
for 50 minutes. Samples were centrifuged at 5000 rpm for 5 minutes
prior to injection. 100 .mu.l injections of each sample were
analyzed in triplicate. The chromatograms were analyzed in GRAMS
software (Thermo Electron Corp., Waltham, Mass.) by integration to
determine areas for respective peaks. Peak area percentages are
calculated based on areas obtained through integrations of SE-HPLC
chromatograms. Peak areas percentages were relative to monomer
control peak areas by the following equation:
Area peak Area monomer control , total .times. 100 ( 1 )
##EQU00003##
Peak area percentages of insoluble aggregates determined by mass
balance:
Area monomer control , total - Area preparation , total Area
monomer control , total .times. 100 ( 2 ) ##EQU00004##
95% confidence intervals were calculated from the triplicate
injections of each sample on the SE-HPLC. SE-HPLC analysis was
performed throughout the course of the study to ensure no changes
in aggregate content.
[0296] Particle Analysis
[0297] Particle analysis was performed using Micro-Flow Imaging.TM.
on a DPA 4100 (Brightwell Technologies, Inc., Ottawa, Ontario,
Canada). Particle free, 0.2 micron filtered water was flushed
through the system prior to sample analysis to obtain a clean
baseline and optimize illumination. Three 0.5 ml samples of each
preparation were analyzed at a flow rate of 0.1 ml/min through a
high magnification flow cell using the "set-point 3" configuration,
which allows detection of particles 1-50 .mu.m. Prior to analysis,
samples were slowly inverted 10 times to ensure suspension of
particles. Negative controls of buffer were also analyzed to
eliminate any buffer influence to particle detection. The data
obtained were number counts per volume per 0.25 micron diameter
size bins. Approximate mass of protein in particles was calculated
by assuming spherical particles with a density of 1.2 g/ml, between
that of protein and water. For ease of data representation, number
counts were summed for sizes 1-5 micron, 5-10 micron, 10-15 micron,
15-20 micron, 20-25 micron, 25-30 micron and 30-50 micron
ranges.
[0298] Infrared Spectroscopy
[0299] Infrared spectra for insoluble aggregates of mGH were
acquired using a Bomem.TM. IR spectrometer (Quebec, Canada) and a
deuterized triglycine sulfate KBr detector. A variable path length
CaF2 cell was used for all the measurements. The solutions were
centrifuged at 1,700 g for 5 minutes and the supernatant removed.
The aggregates were resuspended in buffer to ensure analysis of
aggregates only. Buffer corrections were made by subtracting
spectra of buffer or buffer with protein-free particles. The method
used to obtain and analyze spectra has been described previously
(23). All mathematical manipulations of spectra were performed in
GRAMS software (Thermo Electron Corp., Waltham, Mass.).
[0300] Front-Face Fluorescence
[0301] Front face fluorescence and fluorescence quenching
spectroscopy were used jointly to analyze the tertiary structures
of native, unfolded and adsorbed mGH. Triplicate samples of 3 ml of
each preparation were analyzed in a 3 mm cuvette at a 53.degree.
angle from the excitation beam. An excitation wavelength of 295 nm
was used in a SLM-Aminico Spectrofluorometer (SLMAminico, Urbana,
Ill.). Twelve aliquots of a 7.6 M acrylamide solution were added
until a final acrylamide concentration of 0.4 M was obtained. A
stirrer was used to prevent settling of particles during analysis.
Fluorescence intensities were recorded for 300-380 nm at a scan
rate of 0.95 nm/s. All samples were temperature controlled to
25.degree. C. during analysis. Measured fluorescence emissions were
corrected for dilution and inner filter effects when appropriate.
Buffer corrections were made by subtracting the spectra of buffer
or protein-free particle suspension. Stern-Volmer plots were used
to analyze the data using the relationship:
F 0 F = 1 + K SV [ Q ] ( 3 ) ##EQU00005##
In equation (3), K.sub.sv is the Stern-Volmer constant (M-1).
F.sub.0 and F are fluorescence intensities in the absence and
presence of different concentrations of quencher Q, respectively.
[Q] is the acrylamide concentration (M).
[0302] Immunogenicity Testing in Animals
[0303] All samples were tested in adult .gtoreq.6 weeks of age)
CB6F1 mice for immunogenicity. At the start of the study, Day 0,
blood was obtained retro-venous orbitally from the mice so that
each mouse served as its own baseline. Groups of 8 mice were then
given subcutaneous injections of 2 .mu.g of mGH diluted in saline
for injection five days a week for three weeks (Days 0-4, Days
7-11, Days 14-18). In addition to the mGH samples, one negative
control of buffer alone and a positive control of ovalbumin were
injected in groups of mice. The mice were bled again retro-venous
orbitally on days 7, 21 and 35 and given 5 additional subcutaneous
injections of 2 .mu.g of mGH as a booster on days 35-39. Mice were
bled on days 42 and 49 to monitor any secondary responses.
[0304] The collected sera were tested for IgG1, IgG2a, IgG2b, IgG2c
and IgG3 specific antibody response using an enzyme-linked
immunoassay (ELISA). The wells of Immulon 4 High Binding Affinity
plates (ISC Bioexpress, Kaysville, Utah) were incubated with 100
.mu.l of diluted stock (10 .mu.g/ml) 100 .mu.l of ovalbumin
standard (10 .mu.g/ml) at lab temperature overnight with gentle
agitation. The wells were then drained and washed three times with
PBS containing 0.05% tween 20. After the final wash the wells were
tapped dry on a paper towel. The wells were then blocked with 300
.mu.l of 2% bovine serum albumin (BSA) in PBS for 1 hour. After
application of the blocking solution the wells were washed three
times with a solution of PBS containing 0.05% tween 20. Wells in
rows B-H were then loaded with 50 .mu.l of dilution buffer (1% BSA
in PBS). The sera were then diluted 1:20 into the dilution buffer
and added to the wells in row A. Each plate had two standard curves
of known concentration of mouse monoclonal antibody to ovalbumin
(Abcam ab17291, Cambridge, Mass.). Using a multichannel pipet, 50
.mu.l of the diluted sera from row A were transferred to the wells
in row B. The solution in row B was then mixed by drawing up and
expelling 50 .mu.l (5 times) into the wells before transferring 50
.mu.l to wells in row C. The 2.times. dilutions were continued
through row G. The plates were then sealed and allowed to incubate
at lab temperature for 60 minutes. Then, the wells were washed five
times with a solution of 0.05% tween 20 in PBS and tapped dry on a
paper towel. The wells were incubated with 50 .mu.l of a goat
polyclonal specific to mouse IgG1 (Abcam, ab9165) mouse IgG2a
(Abcam ab9163), mouse IgG2b (Abcam, ab9164), mouse IgG2c (Abcam,
ab9168) or mouse IgG3 (Abcam, ab9166) diluted 1:8000 in dilution
buffer. After 1 hour, the wells were washed five times with 0.05%
tween 20 in PBS and tapped dry on a paper towel. The wells were
subsequently incubated with 50 .mu.l of horse radish peroxidase
conjugated rabbit polyclonal to goat IgG (Abcam, ab6741) diluted
1:5000 in dilution buffer for 60 minutes. The plates were then
washed five times with 0.05% tween 20 in PBS and tapped dry,
followed by the addition of 50 .mu.l of 3,3',5,5'
tetramethylbenzidine to each well. After 20 minutes, 50 .mu.l of
0.5 M sulfuric acid was added to the wells to stop the
reaction.
[0305] The absorbance was recorded with a Molecular Devices
(Sunnyvale, Calif.) "V max" kinetic plate reader at a wavelength of
450 nm and a reference wavelength of 595 nm. The ELISA response was
reported in units of ng/ml and was calculated from the average
absorbance response on a standard curve multiplied by its dilution
factor. The standard curve (r2 value of 0.99) was generated from
the standards on each plate using a four-parameter fit in Softmax
(Sunnyvale, Calif.) with an antibody concentration range of 1250
ng/ml to 10 ng/ml.
[0306] Statistical Analysis of Antibody Responses
[0307] The data were analyzed for statistical differences using a
one tailed t test with variances unknown and not necessarily equal.
An F test was performed to determine that the variances were
different among sample groups. Each group had a sample size of
eight. Microsoft Excel was used to calculate p values. The
probability of a [p] between means of groups was compared with a
90% confidence interval. When comparing means, probabilities of
[p]<0.1 were significant based on the 90% confidence interval
chosen.
Results
[0308] Isotype responses to mGH aggregates. IgG isotypes (IgG1,
IgG2a, IgG2b, IgG2c and IgG3) were assayed by ELISA. No antibodies
against untreated control mGH (stock) were detected until day 21.
Aggregates of mGH most frequently provoked IgG1 isotype responses.
Samples containing mGH adsorbed on glass or alum were the only
samples to break tolerance in all 8 mice in their respective
groups. Injections of glass- and alum-adsorbed samples caused
higher IgG1 responses than all other samples. The IgG1 responses
induced by glass and alum preparations were not different from one
another (p=0.441). The other mGH samples were not able to break
tolerance in all the animals in their group. IgG1 antibody
responses provoked by stock, agitated, HP agitated, freeze-thaw,
and HP freeze-thaw samples are not significantly different from one
another. See FIG. 14.
[0309] The agitated mGH sample caused a higher IgG2a response
compared to all other mGH samples except its high-pressure treated
counterpart. FIG. 15. For all mGH treatments, IgG2b isotype
responses were observed in more mice than were IgG2a or IgG2c
responses. However, mGH adsorbed to glass was the only sample to
produce statistically higher IgG2b responses, and only compared to
stock, HP stock, freeze-thaw and HP freeze-thaw samples. The IgG2c
response caused by the stock preparation was higher than those
resulting from HP stock, freeze-thaw, and HP freeze-thaw samples;
however this statistical difference disappeared by the 6th bleed.
The IgG2c response produced by mGH adsorbed to alum was higher than
responses from HP stock, agitated, freeze-thaw and HP freeze-thaw
and this difference was magnified in the responses measured in the
serum from the 6th bleed (p=0.085). There were no statistical
differences in IgG3 responses between any of the mGH preparations
for the serum samples from bleed 5. FIG. 16.
[0310] Immune Response to High-Pressure Treated Samples.
[0311] Although the stock mGH was immunogenic with 4 out of 8 mice
developing IgG1 antibodies against mGH, the high-pressure treated
stock sample did not break tolerance in any of the 8 mice injected.
In contrast, high-pressure treatment of the freeze-thaw and
agitated samples did not reduce the immune responses induced
against these samples (p=0.133, p=0.378).
[0312] Secondary Responses to mGH Aggregates.
[0313] Administration of mGH adsorbed to alum or glass
microparticles induced the highest levels of IgG1 and also provoked
typical secondary immune responses as evidenced by the
order-of-magnitude higher levels of IgG1 in the 6th bleed compared
to earlier time points. The responses to mGH adsorbed on alum
measured at bleed 6 were significantly higher than those at bleed 5
and bleed 4 (p=0.042, p=0.055). Similarly, the antibody responses
from bleed 6 of mice injected with mGH adsorbed to glass
microparticles were higher than responses at bleed 5 and bleed 4
(p=0.011 and 0.009, respectively). In contrast, although the
antibody responses in the mice injected with high-pressure treated,
freeze-thawed mGH have a qualitatively similar trend to the alum
and glass antibody responses, the response detected at bleed 6 was
not significantly higher than responses at bleed 5 and bleed 4
(p=0.190, p=0.139). Thus, only samples adsorbed on glass or alum
provoked detectable secondary immune responses.
[0314] Mouse Weight Gain.
[0315] The average weight gain of the entire mouse population over
the 7 weeks of the study was 4.5.+-.0.5 grams. No differences in
average weight gain were observed for the different treatment
groups, including mice that were injected with buffer alone
(negative control). Previous studies reported that the average
weight gain from 6 weeks to 13 weeks for CB6F1 female mice is
3.+-.2 grams (24). Therefore, in our study no preparation of mGH
promoted excessive weight gain or weight loss.
[0316] Size-Exclusion Chromatographic Analysis.
[0317] No soluble aggregates were detectable by SE-HPLC in any of
the mGH preparations. The mass loss in the monomeric peak in the
chromatogram was used to determine the percentage of protein in
insoluble aggregates (Tables 1 and 2). The highest levels of
insoluble aggregates were in the freeze-thawed and agitated
samples. After pressurization 100% of the area of the monomer peak
was measured by SE-HPLC. In samples containing glass or alum
microparticles, 100% of the mGH was adsorbed to the microparticles.
Samples with mGH adsorbed to glass or alum were not treated with
high pressure.
[0318] Micro-Flow Imaging Analysis.
[0319] The samples were analyzed for subvisible particles using a
Micro-Flow Imaging.TM. (MFI) instrument. Large numbers of particles
were detected in all samples, including those where aggregates were
undetectable by SE-HPLC analysis (FIG. 10). High-pressure treatment
of the stock, agitated and freeze-thawed samples decreased both the
numbers of particles and the mass of protein estimated to be in the
particles by approximately two orders of magnitude (Table 2). Alum
and glass microparticles were predominately of the 1-1.25 micron
diameter (FIG. 11).
[0320] Analysis of mGH Secondary Structure in Aggregates and
Adsorbed to Glass or Alum.
[0321] Infrared spectra were obtained for mGH in insoluble
aggregates formed during freeze-thawing or agitation and for the
protein adsorbed to microparticles of glass or alum. Due to the
relatively low solubility (ca. 1 mg/ml) of mGH, the stock and high
pressure treated samples could not be concentrated sufficiently to
obtain a high quality spectrum for the monomeric, soluble protein.
Previous studies reported that infrared spectra of protein adsorbed
to alum show native structure (25). Therefore, we assume that mGH
adsorbed to alum has native secondary structure. In previous
circular dichroism analysis of the monomeric mGH we reported that
the protein contained 60% .alpha.-helix (21). In the second
derivative infrared spectrum, a-helix is represented by a strong
negative band at 1654 cm.sup.-1, which is consistent with the
spectrum for the protein adsorbed to alum (FIG. 12). Also, this
spectrum is virtually identical to that for native human growth
hormone (11, 26). mGH adsorbed onto glass microparticles had only a
slight reduction in .alpha.-helix, whereas the protein in insoluble
aggregates induced by freeze-thawing or agitation had reduced (by
ca. 10%) .alpha.-helix content.
[0322] Acrylamide Quenching Fluorescence Spectroscopic Analysis of
Tertiary Structure in Adsorbed mGH.
[0323] We compared the Stern-Volmer constant for the adsorbed
protein to that for native and unfolded mGH. FIG. 13. The
Stern-Volmer constant of native mGH in solution was 0.31.+-.0.17
M-1. Acrylamide has ready access to the tryptophan in unfolded mGH,
as evidenced by a Stern-Volmer constant of 9.4.+-.1.2 M-1. mGH has
one tryptophan (27). Although the high resolution structure of mGH
is not known, the single tryptophan residue in human GH (hGH) is
buried in an .alpha.-helix bundle (28). Due to sequence
similarities between mGH and hGH and our quenching results it is
clear that the tryptophan residue in native mGH also has minimal
solvent exposure. For the mGH adsorbed to alum or glass
microparticles the Stern-Volmer constants were 0.84.+-.0.08 and
1.1.+-.0.2 M.sup.-1 respectively suggesting that the tertiary
structure of mGH adsorbed to glass and alum microparticles is
minimally perturbed from that of native mGH.
Discussion
[0324] Subcutaneous administration of aggregates of mGH broke
immune tolerance to mGH in mice. We characterized the aggregates by
SE-HPLC, spectroscopic analysis and MFI. MFI analysis detected
subvisible protein aggregates (1 to 50 micron) in samples that were
not detectable by SE-HPLC. In addition to particle counts, we
estimated the mass of protein contained in particles in stock, HP
stock, agitated, HP agitated, freeze-thaw and HP freeze-thaw
samples. Aggregate concentrations below 2% can be difficult to
detect and quantify by chromatographic analysis (29). However, MFI
analysis was able to detect aggregates at concentrations as low as
0.001%. For agitated and freeze-thaw preparations of mGH, MFI and
SE-HPLC analyses reported similar percentages of insoluble protein
aggregates.
[0325] Although the stock solution contained particulate aggregate
levels below limits of detection by SE-HPLC, based on the MFI
results it is estimated that the mass of protein in subvisible
particles was equivalent to 1.6 ng aggregate/injection. This level
of particulate aggregate was able to induce immune responses in a
fraction of the mice. High pressure treatment reduced particle
counts by two orders of magnitude to equivalent doses of 0.02 ng
aggregate/injection, which did not break tolerance in any mice.
These results suggest that there may be a threshold level of
aggregate content within a therapeutic formulation above which
immune responses may be generated. Furthermore, they suggest that
subvisible particles of protein may be most potent in eliciting an
immune response.
[0326] In our study, adsorbing mGH to glass or alum yielded a
strong adjuvant response. Not only did the glass and alum samples
induce the highest antibody production, and immune responses in the
most mice, they both provoked secondary immune responses. Vaccine
formulations commonly employ adjuvants to sensitize immune
responses to antigen. Although there is debate about the
mechanism(s) by which aluminum salts increase immune responses to
adsorbed antigens, it has been suggested that aluminum salts and
other particulate adjuvants can enhance uptake by macrophages as
well as induce immunostimulatory cytokines that can lead to faster
maturation of dendritic cells (31). Aluminum hydroxide enhances
immune responses in a T-cell dependent, Th2 biased fashion,
resulting in higher IgG1 than IgG2a isotype titers (32). This is
consistent with our results wherein .mu.g/ml concentrations of
IgG1, but undetectable levels IgG2a, were produced in mice
immunized with mGH adsorbed to alum. Interestingly, administration
of mGH adsorbed to glass microparticles yielded the same result.
This suggests that microscopic glass particles may also induce
T-cell dependent immune responses by enhancing protein uptake by
macrophages. Because microscopic glass contaminants are ubiquitous
in protein solutions stored in glass containers (33), this result
raises the possibility that primary containers may contribute to
immunogenicity of therapeutic proteins.
[0327] Furthermore, generation of IgG1 was most frequently
encountered response to all of the mGH samples tested. IgG1
responses are characteristic of T-cell dependent immune responses
(34). Thus, it appears that subcutaneous injection of all of the
aggregate types of mGH tested in this study induces T-cell
dependent activation of B cells leading to antibody production.
[0328] The only mGH samples that produced both IgG2a and IgG2b
isotypes were those containing agitation-induced aggregates. IgG2
antibodies often are generated in response to viral infections and
lead to cytokine-induced Th1 responses (35-37). Th1 responses
stimulate macrophage rather than B cell activation (38), which
could explain why the agitation-induced aggregates also provoked
the lowest IgG1 concentrations. We speculate that aggregates
produced by agitation may exhibit a relatively ordered and
repetitive surface structure, leading the immune system to
recognize it as a virus.
[0329] Also important to note is that the strain of mice used in
this study was CB6F1, which is the first generation of a cross
between the C57BL/6 and BALB/c strains. The C57BL/6 strain has the
Igh1-b allele which leads to the deletion of the IgG2a isotype
(39). BALB/c mice are known to not produce IgG2c isotype due to the
Igh1-a allele (39). By crossing the two strains we achieve
production of all three IgG2 isotypes. Even though all three
isotypes of IgG2 were detected, the IgG2b isotype responses were
more prevalent than the IgG2a and IgG2c isotype responses.
[0330] The IgG3 isotype is an indicator of T-cell independent
B-cell activation (40-42). Although there were IgG1 and IgG2
isotype responders in each group, the IgG3 isotype was not
significantly produced in any of the groups of mice, consistent
with a T-cell dependent mechanism for breaking tolerance.
[0331] Our results show that even at levels that are below the
limit of detection of SE-HPLC, aggregates of mGH are capable of
inducing potent T cell-dependent responses. These data suggest that
even small fractions of protein aggregates contaminating
therapeutic products can have serious implications. The particles
we injected in mice were predominately of the 1-10 micron in
diameter size ranges. This size range is notoriously difficult to
detect, and is currently not regulated in USP-required tests.
Subvisible particulate aggregates of this size range are most
likely taken up by phagocytes when injected subcutaneously, which
can lead to a strong T-cell dependent immune response taking place
in the lymph nodes. Lymph conduits drain antigens and antigen
presenting cells to lymph nodes and B follicles. These "B-cell
highways", are one pathway by which T-cells and B-cells in the
lymph nodes may encounter antigens (43). Antigens must be less than
70 kDa in size to successfully enter these conduits (43). Larger
particulate antigens, such as those we injected in mice, are
phagocytosed by macrophages or dendritic cells and do not
independently enter lymph conduits. Therefore, most insoluble
protein aggregates are too large to migrate to the lymph node and
activate B-cells independent of T-cells.
[0332] In addition to antigen size, the route of administration can
also have an influence on immunogenicity of a therapeutic protein
(44, 45). Antigens delivered by different routes of administration
may migrate to different organs such as lymph nodes and spleen,
where they may provoke different immune responses. Studies by
Dintzis et al. (46), report that antigens capable of activating B
cells independent of T cells had to be at least 100 kDa in size
when injected intraperitoneally. The mGH aggregates produced in our
study had molecular weights greater than 100 kDa, but were injected
subcutaneously. Subcutaneous injections stimulate activity in the
lymph nodes (47). In contrast, the main lymphoid organ responsible
for antibody production for intraperitoneal injections is the
spleen (47). The peritoneum has a low population of dendritic cells
compared to macrophages and peritoneal macrophages have lower
antigen-presenting capacity (48, 49). Thus, intraperitoneal
injections may expose B-cells to large aggregates independent of
antigen presenting cells. The ability of antigen to activate B
cells independent of T cells may also depend on the population of B
cells in the lymphatic tissue that the immune response takes place.
Lymph nodes have high populations of T cells and low populations of
B cells (50, 51). In contrast, the spleen has high populations of B
cells and lower concentrations of T cells (50, 51). Due to the
greater population of B cells in the spleen, it is more probable
for antigen to encounter B cells and thus activate them without the
assistance of T-cells.
[0333] In conclusion, frequent subcutaneous administrations of even
very low levels of subvisible protein aggregates have the ability
to break tolerance in mice. Furthermore, administration of protein
adsorbed to microparticles can induce potent T-cell dependent
immune responses, much like an adjuvanted vaccine. Clearly, a need
exists for new analytical detection and regulation of subvisible
particles in final product formulation to minimize any potential
adverse effects.
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Example 3
Subvisible Particle Analysis of Betaseron
[0386] The purpose of this example was to examine the presence of
subvisible particles in Betaseron, an Interferon Beta-1 b
formulation manufactured by Bayer.
Materials and Methods
[0387] Betaseron.RTM. (Bayer, Lot#AA8004A) was bought from the
University of Colorado School of Pharmacy Apothecary. Three vials
were reconstituted following the manufacturer's instruction in the
provided diluent (0.54% sodium chloride) and analyzed in duplicate
by MFI. One syringe of diluent solution was recovered to analyze
its particle content.
[0388] Particle analysis was performed using Micro-Flow Imaging.TM.
on a DPA 4100 (Brightwell Technologies, Inc.). 0.2 micron filtered
water was flushed through the system prior to sample analysis to
obtain a clean baseline. Optimize illumination was performed using
Betaseron's diluent solution. Approximately 0.32 ml samples of each
preparation were analyzed at a flow rate of 0.1 ml/min through a
high magnification flow cell using a configuration to detection of
particles 1.125-100 .mu.m. The data obtained were number of
particles per volume per size range.
[0389] Results
[0390] Betaseron is formulated with human albumin (HSA) thus
preventing standard SEC-HPLC chromatography. Studies by Runkel et
al. demonstrate that Betaseron contains approximately 60%
aggregates in higher order aggregates and aggregates complexed with
HSA.
[0391] Betaseron were analyzed by MFI. Three vials were
reconstituted and one syringe of diluents solution was recovered to
analyze its particle content. After Betaseron reconstitution,
approximately 1.2 ml were recovered, enough volume to analyze each
sample in duplicate. When analyzing Betaseron formulations, the
particle counts ranged between 38,000 and 147,000 particles/ml
(FIG. 17) showing the high variability in particle content between
the three preparations; on average Betaseron had 89,800
particles/ml. Particle size distribution is shown in FIG. 18.
Betaseron buffer had very low levels of particle counts indicating
that the protein in the formulation is the main contributor to the
particles detected by MFI. These results suggest that the particles
are caused by aggregation of the protein present in the
formulation.
Example 4
Transgenic Mouse Study--Immunogenicity of IFN-Beta Formulations
[0392] The purpose of this Example was to confirm that aggregates
and subvisible particles present in current Betaseron formulations
leads to the development of binding antibodies to monomeric
IFN-beta. Avonex and pressure treated IFN-beta-1 b were used for
comparison.
[0393] Materials and Methods
[0394] Commercial Betaseron (Lot#WA9497A) and Avonex (Lot#P32033)
formulations were purchased and used as formulated. Pressure
treated IFN-beta-1 b was used at a protein concentration of 0.11
mg/ml, formulated 25 mM acetate (pH 4.0), 9% trehalose, 0.01% Tween
20. SEC-HPLC measurements demonstrated that this material contained
less than 0.05% high molecular weight aggregate.
[0395] Dosing of the mice was performed as described in Hermelling
et al. [1]. Briefly, all animals (5 per group) were dosed 5 mcg
i.p. daily.times.5.times.3 wks. After five days of dosing, two days
were dose free. One group was assigned to pressure treated
IFN-beta-1 b, Betaseron, and Avonex respectively. Blood draws were
taken on time 0, 10, 20, and 26 days and analyzed for the
development of binding antibodies to monomeric IFN-beta-1 b by an
ELISA protocol.
[0396] Results
[0397] Absorbance readings were obtained for each group and
normalized to the response of a non-transgenic mouse dosed with rhl
FN-beta that resulted in a high binding antibody titer. The results
are shown in FIG. 19. Animals dosed with Betaseron developed a
statistically significant (p<0.0001) amount of binding
antibodies to monomeric IFN-beta-1b.
[0398] Conclusion
[0399] Dosing of Betaseron resulted in a significant development of
binding antibodies to monomeric Betaseron relative to baseline
Neither Avonex or pressure treated IFN-beta-1b developed a
significant response. Long-term dosing of Avonex in humans has
demonstrated low-levels of immunogenicity, however with a smaller
fraction of patients developing anti-IFN-beta antibodies relative
to the .about.40% of patients who develop antibodies after
Betaseron dosing.
[0400] The following references are hereby incorporated by
reference.
REFERENCES
[0401] (1) Hermeling, Suzanne. 2005. Structural aspects of the
immunogenicity of therapeutic proteins: transgenic animals as
predictors for breaking immune tolerance/Suzanne Hermeling--[S.I.]:
[s.n.], Tekst.--Proefschrift Universiteit Utrecht. [0402] (2)
Hermeling, Suzanne, W. Jiskoot, D. Crommelin, C. Borns and H.
Schellekens. 2005. Development of a Transgenic Mouse Model Immune
Tolerant for Human Interferon Beta. Pharmaceutical Research Vol.
22(6). [0403] (3) Hermeling S, Schellekens H, Maas C, Gebbink M F B
G, Crommelin D J A, Jiskoot W. (2006) Antibody response to
aggregated human interferon alpha2b in wildtype and transgenic
immune tolerant mice depends on type and level of aggregation. J
Pharm Sci 95: 1084-96.
Example 5
The Use of High Pressure for the Removal of Subvisible Particles
from Etanercept Formulations
Materials and Methods
[0404] Etanercept (50 mg in a SureClick Autoinjector) was diluted
to 10 mg/ml in formulation buffer (25 mM sodium phosphate, 25 mM
L-arginine hydrochloride, 100 mM NaCl, 1% sucrose, pH 6.3) and
analyzed by SEC-HPLC, SDS-PAGE and Micro-Flow Imaging (MFI) before
and after high hydrostatic pressure treatment.
[0405] High Hydrostatic Pressure Treatment was conducted as
follows.
[0406] Experiment 1: Diluted etanercept and formulation buffer were
loaded into sealed syringes prepared to accommodate high pressure
treatment and subject to different high pressures (1000, 1500,
2000, 2500 and 3000 bar) in a PreEMT150.TM. pressure vessel or left
at atmospheric pressure for 16 hr at 25.degree. C. All experiments
were done in singlet. High pressure treated samples were then
depressurized stepwise at a rate of 250 bar/5 min, sealed luer tips
were cut open and samples placed in labeled tubes for further
analysis. Pressure treatment was generated using custom-built, high
pressure vessels as described previously.
[0407] Experiment 2: Diluted etanercept and formulation buffer were
prepared as in experiment 1 in quadruple and subject to 2000 bar or
left at atmospheric pressure for 16 hr at 25.degree. C.
[0408] For Size Exclusion Chromatography (SEC-HPLC), initial and
high pressure treated etanercept (10 mg/ml) were analyzed on a
Tosoh G3000 SWXL using the Agilent 1100 HPLC system in 100 mM NaCl,
100 mM phosphate pH 6.8, at a flow rate of 0.6 ml/min for 35 min
and detected at 280 nm.
[0409] For Micro-Flow Imaging Analysis (MFI), particle analysis was
performed using Micro-Flow Imaging.TM. on a DPA 4100 (Brightwell
Technologies, Inc.). 0.2 micron filtered water was flushed through
the system prior to sample analysis to obtain a clean baseline.
Optimize illumination was performed using formulation buffer. 0.45
ml samples of each preparation were analyzed at a flow rate of 0.1
ml/min through a high magnification flow cell using a configuration
to detection of particles 1.125-50. The data obtained were number
counts per volume per size range.
Results
[0410] The purpose of this example was to examine the presence of
subvisible particles in etanercept formulations and determine if
high pressure treatment could be used to decrease subvisible
particle content. Etanercept, a dimeric fusion protein made up of 2
extracellular domains of the human TNFRII receptor linked to the Fc
portion of a type 1 human immunoglobulin has been shown to cause
2-6% of RA patients to develop anti-entanercept antibodies.
Bressler et al., Optimizing use of tumor necrosis factor inhibitors
in the management of immune-mediated inflammatory diseases, J.
Rheumatol. Suppl. 85:40-52 (2010).
[0411] Commercial prefilled syringes of etanercept formulations
were purchased, diluted to 10 mg/ml using sterile, particle
controlled formulation buffer, and analyzed for aggregate content
using SEC chromatography and micro-flow imaging (FIG. 20). Results
show that etanercept has a 2.2% of aggregate content in its final
formulation as analyzed by SEC-HPLC. When etanercept was analyzed
by MFI, an average of 224,000 particles/ml were detected and the
majority of the particles were in the range of 1-5 .mu.m in size.
SEC is a technique that can only detect particles smaller than 100
nm thus anything larger is undetected. MFI, can detect subvisible
particles in the size range of 1-100 .mu.m and these results
indicate that etanercept has an aggregate content higher than was
described before by other techniques.
[0412] In a preliminary high pressure treatment experiment, diluted
etanercept was pressure treated at 1000, 1500, 2000, 2500, and 3000
bar, 25.degree. C., for 16 hours and reassessed for aggregate
content. FIG. 21 shows no difference between atmospheric and
pressure treated samples as analyzed by SEC-HPLC indicating that
pressure treatment did not alter the content of soluble aggregates
(dimer and larger). When analyzing samples by MFI, the amount of
subvisible particles in etanercept formulations decreased by
approximately 25% at pressures ranging 1000-2000 bar (FIG. 22). At
high pressures (3250 bar) the amount of subvisible particles
increased to .about.290,000 particles/ml. These results show that
the decrease in aggregate content was dependent on pressure
treatment, being most effective at about 2000 bar. Additionally,
the increase of subvisible particle content observed after
application at high pressures of 3000 bar demonstrate the
dependence of the pressure window and that pressure-induced
aggregation can occur for some proteins. In a second experiment,
etanercept diluted formulations (10 mg/ml) were pressure treated at
2000 bar in quadruple and analyzed by MFI (FIG. 23). Results show
that pressure treatment reduced the amount of subvisible particles
by >30%.
* * * * *