U.S. patent application number 13/584631 was filed with the patent office on 2012-12-06 for process for the measurement of the potency of glatiramer acetate.
This patent application is currently assigned to Teva Pharmaceutical Industries, Ltd.. Invention is credited to Ety Klinger.
Application Number | 20120309671 13/584631 |
Document ID | / |
Family ID | 23326090 |
Filed Date | 2012-12-06 |
United States Patent
Application |
20120309671 |
Kind Code |
A1 |
Klinger; Ety |
December 6, 2012 |
PROCESS FOR THE MEASUREMENT OF THE POTENCY OF GLATIRAMER
ACETATE
Abstract
The subject invention provides a process for measuring the
relative potency of a test batch of glatiramer acetate. In
addition, the subject invention provides a process for preparing a
batch of glatiramer acetate as acceptable for pharmaceutical
use.
Inventors: |
Klinger; Ety; (Tel Aviv,
IL) |
Assignee: |
Teva Pharmaceutical Industries,
Ltd.
|
Family ID: |
23326090 |
Appl. No.: |
13/584631 |
Filed: |
August 13, 2012 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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13083112 |
Apr 8, 2011 |
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13584631 |
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12231081 |
Aug 28, 2008 |
7923215 |
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13083112 |
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10313726 |
Dec 4, 2002 |
7429374 |
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12231081 |
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60338767 |
Dec 4, 2001 |
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Current U.S.
Class: |
514/1.1 ;
435/29 |
Current CPC
Class: |
G01N 33/505 20130101;
G01N 2333/55 20130101; A61K 38/02 20130101; G01N 33/5044 20130101;
G01N 2333/555 20130101; G01N 33/5047 20130101; G01N 33/5008
20130101; G01N 2333/52 20130101; G01N 33/502 20130101; A61K 31/198
20130101; A61P 37/00 20180101; G01N 2333/5428 20130101; A61P 25/00
20180101; G01N 2333/5412 20130101; G01N 2333/54 20130101; G01N
2333/57 20130101; G01N 33/6866 20130101; G01N 33/6869 20130101;
G01N 33/5038 20130101 |
Class at
Publication: |
514/1.1 ;
435/29 |
International
Class: |
C12Q 1/02 20060101
C12Q001/02; A61K 38/02 20060101 A61K038/02 |
Claims
1. A process for measuring the potency of a test batch of
glatiramer acetate relative to the known potency of a reference
batch which comprises a. immunizing female (SJLXBALB/C)F1 mice
between 8 and 12 weeks of age with a predetermined amount of
glatiramer acetate from the reference batch. b. preparing a primary
culture of lymph node cells from the mice of step (a) 9-11 days
after immunization; c. separately incubating at least five
reference samples, each of which contains a predetermined number of
cells from the primary culture of step (b) and a predetermined
amount of glatiramer acetate between 1 .mu.g/ml and 25 .mu.g/ml
from a reference batch; d. incubating at least two samples, each of
which contains a predetermined number of cells from the primary
culture of step (b) and a predetermined amount of glatiramer
acetate from the test batch; e. determining for each sample in
steps (c) and (d), the amount of interleukin-2 secreted by the
cells in each sample after 18-21 hours of incubation of such
sample; f. correlating the amounts of interleukin-2 secreted by the
samples incubated with the test batch of glatiramer acetate with
the amounts of interleukin-2 secreted by the samples incubated with
the reference batch of glatiramer acetate so as to determine the
potency of the test batch of glatiramer acetate relative to the
reference batch of glatiramer acetate, wherein in each sample in
steps (c) and (d), the predetermined number of cells is
substantially identical, and wherein for each sample containing a
predetermined amount of glatiramer acetate from the test batch
there is a corresponding reference sample containing a
substantially identical predetermined amount of glatiramer acetate
from the reference batch.
2. The process of claim 1, wherein six reference samples are
separately incubated in step (d).
3. A process for measuring the potency of a test batch of
glatiramer acetate relative to the known potency of a reference
batch which comprises a. immunizing a test mammal with a
predetermined amount of glatiramer acetate from the reference
batch; b. preparing a primary culture of cells from the test mammal
of step (a) at a predetermined time after immunization; c.
separately incubating at least two reference samples, each of which
contains a predetermined number of cells from the primary culture
of step (b) and a predetermined amount of glatiramer acetate from a
reference batch; d. incubating at least two samples, each of which
contains a predetermined number of cells from the primary culture
of step (b) and a predetermined amount of glatiramer acetate from
the test batch; e. determining for each sample in steps (c) and
(d), the amount of a cytokine secreted by the cells in each sample
after a predetermined time period of incubation of such sample; f.
correlating the amounts of the cytokine secreted by the samples
incubated with the test batch of glatiramer acetate with the
amounts of the cytokine secreted by the samples incubated with the
reference batch of glatiramer acetate so as to determine the
potency of the test batch of glatiramer acetate relative to the
reference batch of glatiramer acetate, wherein in each sample in
steps (c) and (d), the predetermined number of cells is
substantially identical, and wherein for each immunization sample
containing a predetermined amount of glatiramer acetate from the
test batch there is a corresponding reference sample containing a
substantially identical predetermined amount of glatiramer acetate
from the reference batch.
4. The process of claim 3, wherein the cytokine is an
interleukin.
5. The process of claim 4, wherein the interleukin is
interleukin-2.
6. The process of claim 4, wherein the interleukin is
interleukin-6.
7. The process of claim 4, wherein the interleukin is
interleukin-10.
8. The process of claim 3, wherein the cytokine is
interferon-gamma.
9. The process of claim 3, wherein the mammal produces T cells
specific to glatiramer acetate reference standard.
10. The process of claim 3, wherein the mammal is a rodent.
11. The process of claim 10, wherein the rodent is a mouse.
12. The process of claim 11, wherein the mouse is a female
(SJLXBALB/C)F1 mouse.
13. The process of claim 3, wherein the mammal is about 8 to about
12 weeks old.
14. The process of claim 3, wherein the cells are lymph node
cells.
15. The process of claim 3, wherein the cells are spleen cells.
16. A process for preparing a batch of glatiramer acetate as
acceptable for pharmaceutical use which comprises a. preparing a
batch of glatiramer acetate; b. measuring the relative potency of
the batch according to the process of claim 1; and c. qualifying
the batch as acceptable for pharmaceutical use if the relative
potency so measured is between 80% and 125% of the reference batch
of glatiramer acetate.
17. A process for preparing glatiramer acetate acceptable for
pharmaceutical use which comprises a. preparing a batch of
glatiramer acetate; b. measuring the relative potency of the batch
according to the process of claim 3; and c. qualifying the batch as
acceptable for pharmaceutical use if the relative potency so
measured is between 80% and 125% of the reference batch of
glatiramer acetate.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/338,767, filed Dec. 4, 2001, the contents of
which are hereby incorporated by reference.
[0002] Throughout this application, various references are cited,
using shortened citations within parentheses. Full citations for
these references can be found at the end of the specification,
immediately preceding the claims. These publications, in their
entireties, are hereby incorporated by reference into the
application to more fully describe the state of the art to which
the invention pertains.
FIELD OF THE INVENTION
[0003] The present invention relates to methods of standardizing
the measurement of the potency of glatiramer acetate based on
specific recognition of the glatiramer acetate by T cells.
BACKGROUND
[0004] It is desirable to standarize the measurement of the potency
of pharmaceutical compositions as there is an optimum potency and
quality of active component that is effective in treating the
disease for which it is administered.
[0005] Glatiramer acetate (GA, also known as Copolymer-1
(Physician's Desk Reference), Copolymer 1, Cop-1 or COPAXONE.RTM.),
is an approved drug for the treatment of multiple sclerosis (MS).
Glatiramer acetate consists of the acetate salts of synthetic
polypeptides, containing four naturally occurring amino acids
(Physician's Desk Reference): L-glutamic acid, L-alanine,
L-tyrosine, nd L-lysine (Physician's Desk Reference) with an
average molar fraction of L-glutamic acid: 0.129-0.153; L-alanine:
0.392-0.462; L-tyrosine: 0.086-0.100; L-lysine: 0.300-0.374,
respectively. The average molecular weight of glatiramer acetate is
4,700-11,000 daltons (Physician's Desk Reference). Chemically,
glatiramer acetate is designated L-glutamic acid polymer with
L-alanine, L-lysine and L-tyrosine, acetate (salt) (Physician's
Desk Reference). Its structural formula is:
(Glu,Ala,Lys,Tyr).sub.x..chi.CH.sub.3COOH
C.sub.H.sub.NO.sub.4.C.sub.3H.sub.7NO.sub.2.C.sub.6H.sub.14N.sub.2O.sub.-
2.C.sub.9H.sub.11NO.sub.3).sub.x..chi.C.sub.2H.sub.4O.sub.2
CAS-147245-92-9
(Physician's Desk Reference). Glatiramer acetate is also written
as: poly[L-Glu.sup.13-15, L-Ala.sup.39-46, L-Tyr.sup.8,9,10,
L-Lys.sup.30-37].nCH.sub.3COOH.
[0006] Glatiramer acetate was shown to suppress experimental
autoimmune encephalomyelitis (EAE)--an experimental model for
multiple sclerosis (MS) in various animal species (Lando et al.,
1979; Aharoni, 1993). Studies of murine EAE suggested that the
protection against EAE is mediated by T cell activity (Aharoni,
1993). This protection from active induction of EAE by mouse spinal
cord homogenate, in which several auto-antigens are involved, could
be adoptively transferred to normal recipients by injection of
glatiramer acetate-specific T suppressor cells (Aharoni, 1993). In
phase III clinical trials, daily subcutaneous injections of
glatiramer acetate were found to slow progression of disability and
reduce the relapse rate in exacerbating-remitting multiple
sclerosis (Johnson, 1987). Processes of manufacturing glatiramer
acetate are described in U.S. Pat. Nos. 3,849,550 and 5,800,808 and
PCT International Publication No. WO 00/05250.
[0007] It is commonly accepted that a high level of antigen
specificity is a feature of T cell activation. The T cells of the
immune system recognize immunogenic peptides complexed to the major
histocompatibility complex (MHC) class II or I molecules, expressed
on antigen presenting cells (APCs). The specificity of antigen
recognition by T cells is defined by several parameters: 1)
affinity of the T cell receptor to the MHC peptide complex; 2)
primary sequence of the antigenic peptide; and 3) synergistic
effects of certain amino acid combinations within the antigenic
peptide. Based on current knowledge on the mechanism of action of
glatiramer acetate, it is believed that the biological activity of
glatiramer acetate in MS is mediated by immunomodulation of T cell
activity.
SUMMARY OF THE INVENTION
[0008] The subject invention provides a process for measuring the
potency of a test batch of glatiramer acetate relative to the known
potency of a reference batch of glatiramer acetate which comprises
[0009] a. immunizing female (SJLXBALB/C)F1 mice between 8 and 12
weeks of age with a predetermined amount of glatiramer acetate from
the reference batch; [0010] b. preparing a primary culture of lymph
node cells from the mice of step (a) 9-11 days after immunization;
[0011] c. separately incubating at least five reference samples,
each of which contains a predetermined number of cells from the
primary culture of step (b) and a predetermined amount of
glatiramer acetate between 1 .mu.g/ml and 25 .mu.g/ml from a
reference batch; [0012] d. incubating at least two samples, each of
which contains a predetermined number of cells from the primary
culture of step (b) and a predetermined amount of glatiramer
acetate from the test batch; [0013] e. determining for each sample
in steps (c) and (d), the amount of interleukin-2 secreted by the
cells in each sample after 18-21 hours of incubation of such
sample; [0014] f. correlating the amounts of interleukin-2 secreted
by the samples incubated with the test batch of glatiramer acetate
with the amounts of interleukin-2 secreted by the samples incubated
with the reference batch of glatiramer acetate so as to determine
the potency of the test batch of glatiramer acetate relative to the
reference batch of glatiramer acetate, [0015] wherein in each
sample in steps (c) and (d), the predetermined number of cells is
substantially identical, and wherein for each sample containing a
predetermined amount of glatiramer acetate from the test batch
there is a corresponding reference sample containing a
substantially identical predetermined amount of glatiramer acetate
from the reference batch.
[0016] The subject invention also provides a process for measuring
the potency of a test batch of glatiramer acetate relative to the
known potency of a reference batch of glatiramer acetate which
comprises [0017] a. immunizing a test mammal with a predetermined
amount of glatiramer acetate from the reference batch; [0018] b.
preparing a primary culture of cells from the test mammal of step
(a) at a predetermined time after immunization; [0019] c.
separately incubating at least two reference samples, each of which
contains a predetermined number of cells from the primary culture
of step (b) and a predetermined amount of glatiramer acetate from a
reference batch; [0020] d. incubating at least two samples, each of
which contains a predetermined number of cells from the primary
culture of step (b) and a predetermined amount of glatiramer
acetate from the test batch; [0021] e. determining for each sample
in steps (c) and (d), the amount of a cytokine secreted by the
cells in each sample after a predetermined time period of
incubation of such sample; [0022] f. correlating the amounts of the
cytokine secreted by the samples incubated with the test batch of
glatiramer acetate with the amounts of the cytokine secreted by the
samples incubated with the reference batch of glatiramer acetate so
as to determine the potency of the test batch of glatiramer acetate
relative to the reference batch of glatiramer acetate, [0023]
wherein in each sample in steps (c) and (d), the predetermined
number of cells is substantially identical, and wherein for each
immunization sample containing a predetermined amount of glatiramer
acetate from the test batch there is a corresponding reference
sample containing a substantially identical predetermined amount of
glatiramer acetate from the reference batch.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1: Immunization with GA RS (Reference Standard).
Primary culture of LN cells. IL-2 Detection by ELISA.
[0025] FIG. 2: Induction of GA-specific T cells. Primary cultures
of LN cells derived from mice immunized with 250 .mu.g GA RS+CFA or
with CFA alone were cultured in the presence of increasing
concentrations of GA RS. Following overnight incubation at
37.degree. C. the culture media were collected and assayed for IL-2
by ELISA.
[0026] FIG. 3: Diagram of hemacytometer.
[0027] FIG. 4: Optimization of the immunization protocol--culture
source and GA Reference Standard (RS) dose effect. Primary cultures
of Lymph Node (LN) and spleen cells were derived from mice
immunized with 10 or 250 .mu.g GA RS+complete Freund's adjuvant
(CFA). The cells were cultured in the presence of increasing
concentrations of GA RS. Following overnight incubation at
37.degree. C., the culture media were collected and assayed for
IL-2 by enzyme-linked immunoabsorbent assay (ELISA).
[0028] FIG. 5: Optimization of the immunization protocol--adjuvant
and dose effect. Primary cultures of LN cells derived from mice
immunized with 250 .mu.g GA RS+CFA or with 10 mg GA RS+incomplete
Freund's adjuvant (ICFA) were cultured in the presence of
increasing concentrations of GA RS. Following overnight incubation
at 37.degree. C., the culture media were collected and assayed for
interleukin-2 (IL-2) by ELISA.
[0029] FIG. 6: Effect of the immunization period. Mice were
immunized with 250 .mu.g GA RS in CFA and LN were removed after 9,
10 and 11 days. The response of the LN cells from different groups
to various concentrations of GA RS was tested in vitro by measuring
IL-2 accretion by ELISA.
[0030] FIG. 7: Effect of the culture media on GA-specific T cell
response. Primary cultures of LN cells were cultured with different
media containing either 1% normal mouse serum (NMS), 1% fetal
bovine serum (FBS) or defined cell culture media (DCCM1) The cells
were incubated with increasing concentrations of GA RS for 21 hours
at 37.degree. C. Subsequently, the culture media were collected and
assayed for IL-2 by ELISA.
[0031] FIG. 8: Kinetics of IL-2 secretion in response to GA RS. A
primary culture of LN cells was prepared from mice immunized with
250 .mu.g GA RS+CFA. The cells were incubated with 0, 0.5, 2.5 and
5 .mu.g/ml GA RS at 37.degree. C. for the indicated intervals. At
each time point, an aliquot of 5.times.10.sup.6 cells was
centrifuged and the supernatant was kept at -20.degree. C. All
samples were assayed simultaneously for IL-2 by ELISA.
[0032] FIG. 9: Stability of IL-2 in culture media. A primary
culture of LN cells was prepared from mice immunized with 250 .mu.g
GA RS+CFA. The cells were incubated with various concentrations of
GA RS at 37.degree.. After overnight incubation, the supernatants
were collected and divided into two aliquots. One aliquot was
assayed immediately by ELISA, and the second was kept for 7 days at
-20.degree. C. prior to being assayed.
[0033] FIG. 10: Stability of GA RS solution at -20.degree. C. GA RS
solution of 1 mg/ml was prepared, divided into aliquots and kept at
-20.degree. C. The dose-response of the GA-specific cells to GA RS
solution was tested at time zero (Date 1) and after 5 months at
-20.degree. C.
[0034] FIG. 11: Effect of the average molecular weight (MW) on the
GA RS-specific T-cell response. A primary culture of LN cells was
prepared from mice immunized with 250 .mu.g GA RS+CFA. The cells
were cultured in the presence of 2 different concentrations of GA
RS and of GA Drug Substance (DS) of different molecular weights.
Following overnight incubation at 37.degree. C. the culture media
were collected and assayed for IL-2 by ELISA.
[0035] FIGS. 12(A & B): Cross-reactivity of GA RS-specific T
cells with GA DS and Drug Product (DP) batches. A primary culture
of LN cells was prepared from mice immunized with 250 .mu.g GA
RS+CFA. The response of the GA RS-specific T cells to another GA DS
batch (FIG. 12A), to a GA DP batch and to mannitol (FIG. 12B) was
compared to the response to the GA RS batch. IL-2 levels in the
culture media were measured by ELISA.
[0036] FIG. 13: Kinetics of GA RS proteolysis by trypsin. GA RS was
proteolysed by trypsin for the indicated time points. The activity
of the proteolysed samples was tested by the in vitro potency
test.
[0037] FIG. 14: Reverse-Phase High Pressure Liquid Chromatography
(RP-HPLC) of GA before and after proteolysis by trypsin. GA RS was
proteolysed by trypsin and the chromatographic profile of the
samples was tested by RP-HPLC.
[0038] FIG. 15: Kinetics of GA RS proteolysis by chymotrypsin. GA
RS was proteolysed by chymotrypsin for the indicated time points.
The activity of the proteolysed samples was tested by the in vitro
potency test.
[0039] FIG. 16: RP-HPLC of GA before and after proteolysis by
chymotrypsin. GA RS was proteolysed by chymotrypsin and the
chromatographic profile of the samples was tested by RP-HPLC.
DETAILED DESCRIPTION
[0040] The subject invention provides a process for measuring the
potency of a test batch of glatiramer acetate relative to the known
potency of a reference batch of glatiramer acetate which comprises
[0041] a. immunizing female (SJLXBALB/C)F1 mice between 8 and 12
weeks of age with a predetermined amount of glatiramer acetate from
the reference batch. [0042] b. preparing a primary culture of lymph
node cells from the mice of step (a) 9-11 days after immunization;
[0043] c. separately incubating at least five reference samples,
each of which contains a predetermined number of cells from the
primary culture of step (b) and a predetermined amount of
glatiramer acetate between 1 .mu.g/ml and 25 .mu.g/ml from a
reference batch; [0044] d. incubating at least two samples, each of
which contains a predetermined number of cells from the primary
culture of step (b) and a predetermined amount of glatiramer
acetate from the test batch; [0045] e. determining for each sample
in steps (c) and (d), the amount of interleukin-2 secreted by the
cells in each sample after 18-21 hours of incubation of such
sample; [0046] f. correlating the amounts of interleukin-2 secreted
by the samples incubated with the test batch of glatiramer acetate
with the amounts of interleukin-2 secreted by the samples incubated
with the reference batch of glatiramer acetate so as to determine
the potency of the test batch of glatiramer acetate relative to the
reference batch of glatiramer acetate, [0047] wherein in each
sample in steps (c) and (d), the predetermined number of cells is
substantially identical, and wherein for each sample containing a
predetermined amount of glatiramer acetate from the test batch
there is a corresponding reference sample containing a
substantially identical predetermined amount of glatiramer acetate
from the reference batch.
[0048] In one embodiment, six reference samples are separately
incubated in step (d).
[0049] The subject invention also provides a process for measuring
the potency of a test batch of glatiramer acetate relative to the
known potency of a reference batch of glatiramer acetate which
comprises [0050] a. immunizing a test mammal with a predetermined
amount of glatiramer acetate from the reference batch; [0051] b.
preparing a primary culture of cells from the test mammal of step
(a) at a predetermined time after immunization; [0052] c.
separately incubating at least two reference samples, each of which
contains a predetermined number of cells from the primary culture
of step (b) and a predetermined amount of glatiramer acetate from a
reference batch; [0053] d. incubating at least two samples, each of
which contains a predetermined number of cells from the primary
culture of step (b) and a predetermined amount of glatiramer
acetate from the test batch; [0054] e. determining for each sample
in steps (c) and (d), the amount of a cytokine secreted by the
cells in each sample after a predetermined time period of
incubation of such sample; [0055] f. correlating the amounts of the
cytokine secreted by the samples incubated with the test batch of
glatiramer acetate with the amounts of the cytokine secreted by the
samples incubated with the reference batch of glatiramer acetate so
as to determine the potency of the test batch of glatiramer acetate
relative to the reference batch of glatiramer acetate, [0056]
wherein in each sample in steps (c) and (d), the predetermined
number of cells is substantially identical, and wherein for each
immunization sample containing a predetermined amount of glatiramer
acetate from the test batch there is a corresponding reference
sample containing a substantially identical predetermined amount of
glatiramer acetate from the reference batch.
[0057] In one embodiment, the cytokine is an interleukin.
[0058] In a preferred embodiment, the interleukin is
interleukin-2.
[0059] In another embodiment, the interleukin is interleukin-6.
[0060] In a further embodiment, the interleukin is
interleukin-10.
[0061] In an added embodiment, the cytokine is
interferon-gamma.
[0062] In one embodiment, the mammal produces T cells specific to
glatiramer acetate reference standard.
[0063] In another embodiment, the mammal is a rodent.
[0064] In still another embodiment, the rodent is a mouse.
[0065] In an additional embodiment, the mouse is a female
(SJLXBALB/C)F1 mouse.
[0066] In a further embodiment, the mammal is about 8 to about 12
weeks old.
[0067] In yet another embodiment, the cells are lymph node
cells.
[0068] In one embodiment, the cells are spleen cells.
[0069] The subject invention further provides a process for
preparing a batch of glatiramer acetate as acceptable for
pharmaceutical use which comprises [0070] a. preparing a batch of
glatiramer acetate; [0071] b. measuring the relative potency of the
batch according to the process of claim 1; and [0072] c. qualifying
the batch as acceptable for pharmaceutical use if the relative
potency so measured is between 80% and 125% of the reference batch
of glatiramer acetate.
[0073] Additionally, the subject invention provides a process for
preparing glatiramer acetate acceptable for pharmaceutical use
which comprises [0074] a. preparing a batch of glatiramer acetate;
[0075] b. measuring the relative potency of the batch according to
the process of claim 3; and [0076] c. qualifying the batch as
acceptable for pharmaceutical use if the relative potency so
measured is between SOW and 125% of the reference batch of
glatiramer acetate.
[0077] Thus, rte present invention provides the standardization of
the measurement of the potency of GA. The potency test
quantitatively determines the biological activity of GA. This is
the first showing ever of such a test. This standardization method
is essential in order to show batch to batch reproducibility with
regards to potency and quality of DS and DP. In the context of this
application, OS refers to the active ingredient, i.e., GA. DP is
used to indicate the finished product, i.e., Copaxone.RTM.. RS
denotes a batch of glatiramer acetate having an average molecular
weight of about 7000 Da.
[0078] The subject invention makes use of the observation that T
cells incubated with a cytokine, e.g., IL-2, proliferate in
response to that cytokine (Lisak et al., 1974).
[0079] The examples which follow describe the invention in detail
with respect to showing how certain specific representative
embodiments thereof can be made, the materials, apparatus and
process steps being understood as examples that are intended to be
illustrative only. In particular, the invention is not intended to
be limited to the methods, materials, conditions, process
parameters, apparatus and the like specifically recited herein.
EXPERIMENTAL EXAMPLES
General Procedure Outline
[0080] Mice were immunized with 250 .mu.g GA RS in CFA. GA RS was
produced as described in U.S. Pat. No. 5,800,808 or PCT
International Publication No. WO 00/05250. The GA RS was chosen
based on the chemical and biological properties being in the
midrange of Copaxone.RTM. as described above. After 9-11 days, a
primary culture of LN cells was prepared, and the cells were
incubated with various concentrations of GA RS and with test
samples. Following 18-21 hours of incubation at 37.degree. C. in a
humidified CO.sub.2 incubator, the culture media were collected and
the level of IL-2 was measured by ELISA. The T-cell response to
each DS batch were tested at two concentrations (within the linear
range), and the % potency of the DS batch was calculated relative
to that of the GA RS batch.
Example 1
Standard Procedure
Purpose
[0081] The purpose of this procedure was to determine the relative
potency of GA DS batch in vitro, using GA RS-specific T cells.
Equipment
[0082] Laminar hood, hemacytometer, disposable cover slips, cell
counter centrifuge, temperature-controlled shaking incubator,
humidified, temperature controlled 5% CO.sub.2 incubator, light and
inverted microscopes, ELISA reader (450 nm filter), freezer,
refrigerator scissors, forceps, stepper, pipettman 40-200 .mu.l,
pipettman 200-1000 .mu.l, pipettman 5-40 .mu.l, powerpette, sterile
glass syringes and luer bridges.
Disposables
[0083] Cryotubes, 96-well enhanced binding ELISA plate (Nunc, Cat.
#442404), 96-well non-sterile microtest plate (Falcon Cat. #3911),
24-well flat bottom steriled tissue culture plate (Nunc, Cat.
#143982), petri dishes, Eppendorf tubes (polypropylene), steriled
pipette tips 200-1000 .mu.l, pipettes: 2, 5 & 10 ml, laboratory
coat, gloves, 0.2.mu. cellulose acetate filter, filtered system 200
ml (Corning, Cat. #430767), Kim wipes, support platform, 10 ml
syringes, 21Gxl 1/2'' needles, insulin syringes and combitips 5
ml.
Materials and Reagents
For the Immunization Procedure
[0084] 95% ethanol (Bio Lab, Cat. #13680605, or equivalent), 70%
ethanol prepared from 95% ethanol by dilution with distilled water,
phosphate buffered saline (PBS).times.1 (SIGMA, Cat. #3813, or
equivalent), CFA containing 1 mg mycobacterium tuberculosis (MT)
(H37Ra, ATCC 255177), (SIGMA, Cat. #F-5881, or equivalent), and GA
RS batch.
For the In Vitro Bioassay Procedure
[0085] 95% ethanol (Bio Lab, Cat. #13680605, or equivalent), 70%
ethanol prepared from 95% ethanol by dilution with distilled water,
trypan blue (BDH, Cat. #3407), DCCM1 (Defined Cell Culture Media)
(Beit Haemek, Cat. #05-010-1A or equivalent), RPMI 1640 (Roswell
Park Memorial Institute) (Beit Haemek, Cat. #01-100-1A), steriled
L-glutamine 2 mM.times.100 (Bio Lab, Cat. #13.015), steriled MEM
(Minimum Essential Media)--non-essential amino acids.times.100 (Bio
Lab, Cat. #11.080), steriled sodium pyruvate 1 mM.times.100 (Bio
Lab, Cat. #13.016), antibiotic/antimycotic Solution 1 (Bio Lab,
Cat. #13.020), 2-mercaptoethanol (SIGMA, Cat. #M-7154), PBS (SIGMA,
Cat. #3813), concavalin A (Con A) (SIGMA, Cat. #C-5275), MBP
(Myelin Basic Protein) peptide (87-99) (BACHEM, Cat. #H-1964, or
equivalent), and GA RS.
[0086] IL-2 was measured by ELISA kit: OptEIA.TM. Set: mouse IL-2
(Pharmingen, Cat. #2614KI, or equivalent).
Animals
[0087] Female (SJLXBALB/C)F1 mice between 8-12 weeks old (Jackson
Laboratories, Bar Harbor, Me.) were used, although female
(BALB/C)F1 mice between 8-12 weeks old from other sources may be
used. Animal housing and care conditions were maintained in
specific pathogen-free (SPF) conditions.
Solutions
TABLE-US-00001 [0088] TABLE 1 Procedures for making solutions
Steriled PBS The content of one package of PBS was dissolved in 1
liter double distilled water (ddH.sub.2O). The buffer was filtered
through a 0.2.mu. cellulose acetate filter and kept in refrigerator
(2-8.degree. C.) up to one week. 2% (w/v) Trypan blue in About 0.5
g of Trypan blue were dissolved in 25 ml PBS filtered PBS and
stored in refrigerator up to 6 months. 0.1% (v/v) Trypan blue in
0.1% Trypan blue solution was prepared from the 2% PBS Trypan blue
stock solution and filtered through a 0.2.mu. cellulose acetate
filter. The 0.1% Trypan blue solution was stored at room
temperature for up to one month. Steriled 2- 10 .mu.l of
2-mercaptoethanol were added into 9.99 ml Mercaptoethanol of
sterilized PBS and filtered through a 0.2.mu. cellulose acetate
membrane and kept in refrigerator for up to 3 months. GA RS About
10 mg of glatiramer acetate RS were weighed stock solution of
accurately and dissolved in ddH.sub.2O to a concentration 1 mg/ml
of approx and 1.2 mg/ml. The optical density (OD) of the RS
solution was measured at 275 nm. The OD was adjusted to approx.
1.03 with ddH.sub.2O, and a stock solution of 1 mg/ml of GA RS was
obtained. The solution was mixed well, divided into working
aliquots (200-500 .mu.l) and stored at -20.degree. C. until use.
Steriled MEM .times. 100 The steriled solution was divided into
aliquots of 5 ml each, and kept at -20.degree. C. until use. After
thawing, the working aliquot was kept in refrigerator for up to one
month. Antibiotic/ The original 20 ml package was kept at
20.degree. C. The antimycotic package was opened and all the
contents were solution 1 divided into aliquots of 2 ml each and
stored at -20.degree. C. until use. The working aliquot was stable
and was able to be subjected to several freeze-thaw cycles.
Steriled The contents of the opened package were divided
L-glutamine into aliquots of 2 ml each and kept at -20.degree. C.
until 2 mM .times. 100 use. Enriched DCCM1 For 100 ml of sterile
enriched DCCM1, the following medium components were mixed
together: 1 ml of L-glutamine (2 mM), 1 ml MEM, 1 ml sodium
pyruvate (1 mM), 200 .mu.l antibiotic/antimycotic solution 1, 400
.mu.l 2-mercaptoethanol and 96.4 ml DCCM1. The enriched DCCM1 was
filtered through a 0.2.mu. cellulose acetate filter and stored in
refrigerator for up to 1 week. MBP peptide Primary bank: The stock
solution of 10 mg/ml in ddH.sub.2O was prepared, divided into
working aliquots and kept at 20.degree. C. Secondary bank: One
aliquot of the primary bank (10 mg/ml) was thawed and diluted to 1
mg/ml with ddH.sub.2O. The primary bank solution was divided into
working aliquots of 50 .mu.l each and kept at -20.degree. C. Upon
use, aliquot from the secondary bank was thawed and diluted with
enriched DCCM1 to obtain a solution of 20 .mu.g/ml. Con A solution
Primary bank: The contents of one vial of 5 mg Con were dissolved
in 1 ml PBS, mixed well and divided into aliquots of 50 .mu.l each.
The aliquots were kept at -20.degree. C. up to the expiration date
set by the manufacturer. Secondary bank: One aliquot of the primary
bank (5 mg/ml) was thawed and diluted with 4.95 ml of enriched
DCCM1 medium to obtain a solution of 50 .mu.g/ml. The solution was
divided into working aliquots of 100 .mu.l each and kept at
-20.degree. C. Upon use, one aliquot from the secondary bank was
thawed and diluted up to 1 ml (a 10-fold dilution) with enriched
DCCM1 to obtain a solution of 5 .mu.g/ml Con A.
Immunization
[0089] GA RS emulsion in CFA was prepared under sterile conditions,
i.e., in a laminar hood, using sterile equipment and materials.
Preparation of GA RS Solution
[0090] About 15 mg of GA RS were weighed accurately and dissolved
in sterile PBS to a concentration of 5 mg/ml.
Preparation of GA RS Emulsion
[0091] Equal volumes of GA RS solution (5 mg/ml) and CFA were
mixed. The mixture was transferred into a sterile glass syringe
connected to a second glass syringe through a luer bridge. The
mixture was mixed well by being transferred from one syringe to
another until the mixture was well emulsified. A stable emulsion
was confirmed when a drop of the emulsion floated on water without
dispersing.
Injection
[0092] The GA RS emulsion was transferred into an insulin syringe.
Then, 100 .mu.l of the emulsion (250 .mu.g GA per mouse) were
injected into four footpads of each naive mouse (about 25 .mu.l
into each footpad). The immunized mice were used for the in vitro
test 9-11 days following immunization.
Preparation of a Primary Culture of LN Cells
[0093] The primary culture of LN cells was prepared 9-11 days
following immunization, according to the following procedure:
Surgical procedure for Removal of LN Cells
[0094] The UV lamp was turned on 20 minutes before commencing work
in the laminar hood and turned off when work began. Prior to
placing any reagents under the hood, the working surface was
cleaned with a 70% ethanol solution. Enriched DCCM1 medium was
prepared. The enriched DCCM1 and the RPMI medium were pre-warmed at
37.degree. C. prior to use. The mice were sacrificed by cervical
dislocation. Each mouse was placed on its back and fastened to a
support platform. The abdomen was sprayed with 70% alcohol and a
middle incision was made (a 2 cm-long incision was usually
sufficient). The skin was intersected towards the hind legs and LN
was located from the hind and forelegs. The LN was transferred into
a sterile petri dish containing about 5 ml sterile RPMI medium and
the LN cells were teased out by a sterile syringe plunger. The
sterile syringe was used to collect the cells' suspension from the
petri dish (the collection of tissue debris was avoided by using
sterile needles). The cells' suspension were transferred into a 50
ml sterile tube.
Cell Counting: Example Procedure for Counting LN Cells Derived from
5 Immunized Mice
[0095] The cells' tube were filled with RPMI medium up to 40 ml.
The LN cells were centrifuged at 200.times.g for 10 minutes at room
temperature (15-25.degree. C.). The pellet was re-suspended with 40
ml RPMI. Two aliquots of 50 .mu.l were drawn each from the cells'
suspension diluted 4-fold with 150 .mu.l of 0.1% Trypan blue in a
microtest well. The aliquots were mixed well by pipetting gently up
and down. The hemacytometer was covered with a cover slip. A 50-200
.mu.l pipettman was used to load both aliquots into the upper and
lower chambers of the hemacytometer, one suspension in each
chamber. The mixture was allowed to settle within the chambers for
about 2 minutes. Care was taken to not introduce bubbles into the
chamber. The mixture (cell suspension+Trypan blue) was allowed to
cover the entire surface of the chamber. If bubbles were present in
the chamber, or if it was overloaded, the hemacytometer was cleaned
completely and dried with wipes and the chambers were reloaded.
[0096] The viable cells were counted in the central square
(composed of 25 large squares of 16 small squares each, see FIG. 3)
of the upper and lower chambers. The viable cells did not absorb
Trypan blue and were therefore characterized by a clear appearance.
However, dead cells were permeable to Trypan blue and appeared blue
in color. Cells appearing on the border of the central square were
only counted if a portion of the cell was actually within the
central square. If a cell was on the border, and not at all within
the central square, it was not counted. The cells' density was
calculated using the following equation:
Average number viable cells ( both chambers ) .times. 4 .times. 10
4 = cells ml . ##EQU00001##
[0097] The cells were centrifuged at 200.times.g for 10 minutes at
room temperature. The cells were re-suspended to a density of
1.times.10.sup.7 cells/ml with enriched DCCM1.
In Vitro Bioassay
[0098] The in vitro bioassay was performed in a 24-well,
flat-bottomed tissue culture test plate at a final volume of 1
ml.
Preparation of GA RS Calibration Curve
[0099] One aliquot of the 1 mg/ml GA RS stock was thawed. The GA RS
stock solution was diluted to 100 .mu.g/ml (10-fold) with enriched
DCCM1 medium and filtered through a 0.2.mu. cellulose acetate
filter. Six serial dilutions of the GA RS solution with enriched
DCCM1 medium were prepared between 2-50 .mu.g/ml, as described by
the example in Table 2.
TABLE-US-00002 TABLE 2 Example for preparation of GA RS dilutions
GA RS VOLUME (.mu.l) OF GA RS VOLUME CONCENTRATION STOCK SOLUTION
(.mu.l) OF ENRICHED (.mu.g/ml) (100 .mu.g/ml) DCCM1 50 1000 1000 30
600 1400 20 400 1600 10 200 1800 5 100 1900 2 40 1960
Preparation of GA DS Dilutions
[0100] About 10-20 mg of GA DS from the batch to be tested was
weighed accurately and dissolved with ddH.sub.2O to 1.2 mg/ml. The
OD minus blank of the solution was measured at 275 nm. The OD of
the sample was adjusted to approx. 1.03 with ddH.sub.2O to obtain a
stock solution of 1 mg/ml of GA. The stock solution of 100 .mu.g/ml
was prepared with enriched DCCM1 and filtered through a 0.2.mu.
cellulose acetate filter. The stock solution was diluted to 10 and
20 .mu.g/ml as described in Table 2 for the RS batch.
Assay Reaction
[0101] The following were added to the 24-well flat-bottomed tissue
culture plate (see an example of a plate template below):
GA RS
[0102] 0.5 ml of LN cells (final density, for example,
5.times.10.sup.6 cells/well).
[0103] 0.5 ml of each GA RS dilution, thus the final concentrations
of GA RS in the wells were 25, 15, 10, 5, 2.5 and 1 .mu.g/ml.
GA DS samples
[0104] 0.5 ml of LN cells (final density, for example,
5.times.10.sup.6 cells/well).
[0105] 0.5 ml of each sample dilution, thus the final
concentrations of the test sample in the well were 5 and 10
.mu.g/ml.
[0106] Each test included the following controls: [0107] 1)
Negative control--LN cells incubated with a control peptide: [0108]
0.5 ml of LN cells (final density 5.times.10.sup.6 cells/well)
[0109] 0.5 ml of MBP peptide solution (20 .mu.g/ml) in enriched
[0110] DCCM1 (final concentration 10 .mu.g/ml) [0111] 2) Positive
control--LN cells stimulated with Con A (non-specific T cell
stimulant): [0112] 0.5 ml of LN cells (final density
5.times.10.sup.6 cells/well) [0113] 0.5 ml of Con A (5 .mu.g/ml) in
enriched DCCM1 (final concentration 2.5 .mu.g/ml)
Example for a Plate Template
TABLE-US-00003 [0114] GA RS* GA RS* GA RS* GA RS* GA RS* GA RS* 1
.mu.g/ml 2.5 .mu.g/ml 5 .mu.g/ml 10 .mu.g/ml 15 .mu.g/ml 25
.mu.g/ml Sample 1 Sample 1 5 .mu.g/ml 10 .mu.g/ml Sample 2 Sample 2
5 .mu.g/ml 10 .mu.g/ml Sample 3 Sample 3 Negative Positive 5
.mu.g/ml 10 .mu.g/ml Control Control GA RS*--Glatiramer acetate
reference standard.
[0115] The density of the cells was changed depending upon their
response to GA. The cultures were kept at 37.degree. C. in a
humidified 5% CO.sub.2 incubator for 18-21 hrs. The plate was
centrifuged at 200.times.g for 10 minutes at room temperature. The
supernatants were collected into cryotubes. The supernatants were
divided into working aliquots to avoid repeated freezing/thawing of
the samples. The supernatants were stored at -20.degree. C. for up
to one week. The hood was cleaned with 70% ethanol solution and
dried with Kim wipes. The gloves were removed and the hands were
immediately washed with disinfectant.
ELISA for IL-2 Detection
[0116] All samples were tested in triplicate. Each plate run
included the following: [0117] 1) IL-2 standard curve--including at
least 6 non-zero concentrations of IL-2. [0118] 2) Blank [0119]
+1.sup.st antibody, without IL-2 standard, +2.sup.nd antibody (zero
point). [0120] 3) Samples [0121] The culture media of GA RS, test
samples and controls were diluted with enriched DCCM1 as follows:
[0122] a) A 2-fold dilution of the 1 and 2.5 .mu.g/ml GA RS sample
and of the negative control sample; [0123] b) 5-10 fold dilutions
of the 5-25 .mu.g/ml GA RS samples and of the test samples; and
[0124] c) 15-20 fold dilution of the positive control sample (Con
A).
[0125] The ELISA protocol for measuring IL-2 levels was performed
according to the manufacturer's recommendations. If the optical
density of any sample reached the upper/lower limits of the plate
reader, the sample was re-analyzed at a higher/lower dilution,
respectively.
Calculation, and Acceptance Criteria
ELISA Measurements
[0126] The mean absorbance was subtracted of the blank sample (zero
IL-2 standard point) from the absorbance of standards, samples and
controls and calculated for each set of triplicate the mean
(absorbance-blank), standard deviation (SD), and relative standard
deviation (RSD).
Sample Replicates
[0127] Whenever there was a suspected outlier, it was necessary to
ensure that the outlier was statistically based, in order to
elucidate any potential problems that may have affected the overall
results. If the RSD between triplicate measures was higher than 10%
and the average OD-blank was >0.300, outlier rejection was
applied using the Dixon Q-Test, The Dixon Q-Test was used to reject
possible outliers when the relevant acceptance criteria was not
satisfied in a test based on replicates. The outlier test was
applicable only to replicate measurements of the same standard
solution. For less than 10 observations, only 1 outlier was able to
be determined and eliminated. This procedure expanded the use of
the Dixon Q-Test in rejecting outliers from any number of replicate
measurements between 3 and 7, with a confidence level of 95%.
Procedure
[0128] The suspected outlier was designated X.sub.1. All other
measurements were labeled in reference to the suspected outlier,
e.g., X.sub.2 was the value next to the suspected outlier, X.sub.3
was second value from the suspected outlier, X.sub.k was the
farthest from the suspected outlier and X.sub.k-1 was the value
second from the farthest, etc. For 3-7 replicates, the following
equation was used:
X 2 - X 1 X k - X 1 . ##EQU00002##
The appropriate k value was determined from the calculated fraction
using Table 3.
TABLE-US-00004 TABLE 3 k Value No. of Observations (k) Value at
P.sub.95 3 0.94 4 0.76 5 0.642 6 0.560 7 0.507
[0129] If no outlier was identified by the Dixon Q-Test but the %
difference between 2 out of the 3 replicates was not more than 10%,
the closest 2 replicates were used for calculating the % potency.
Otherwise, the ELISA test was repeated for this sample.
[0130] Outlier rejection from samples with OD<0.300 (blank,
negative control and low standard points) was applied. When an
outlier was located, when it was rejected and reported. Duplicate
measures were used for the calculation of % potency.
Blank Samples
[0131] The absorbance of each of the blank samples was .ltoreq.10%
of the mean absorbance of the highest concentration of the IL-2
standard. If one of the blank replicates was beyond the above
limits, it was rejected and duplicate samples were used.
IL-2 Standard Curve
[0132] The IL-2 standard curve was graphed according to the
manufacturer's recommendations. IL-2 standards that exhibit poor
sensitivity, or sample processing error were able to be rejected if
a minimum of six non-zero concentration IL-2 standards remained in
the curve. The back-calculated standard concentration had a
relative error (RE) greater than 20% for the lower calibration
point and .+-.15% for all other concentrations. The IL-2
calibration curve was constructed from at least six non-zero
concentration points (at least 17 calibration points), covering the
range of expected concentrations. The standard curve range was able
to be truncated if the high or low concentrations failed. The
R.sup.2 of the linear regression curve was 0.97.
Assay Controls
[0133] The concentration of IL-2 was calculated in all samples from
the linear regression plot of the IL-2 standard, utilizing the
equation of the linear regression curve. The final concentration of
IL-2 was calculated in all samples by multiplying by the samples'
dilution factor.
Negative Control (MBP Peptide)
[0134] The final concentration of IL-2 in at least 2 out of the 3
replicates of the negative control sample was below the levels of
IL-2 measured for the lowest calibration point of the GA RS
curve.
Positive Control (Con A)
[0135] The final concentration of IL-2 in at least 2 out of the 3
replicates of the positive control sample was similar to or above
the level of IL-2 in the highest calibration point of the GA RS
curve.
Calculation of the Relative Potency of GA DS Batches
GA RS Curve
[0136] The GA RS curve was plotted on a log-log scale, with log
IL-2 concentration on the y-axis and log GA RS concentration on the
x-axis. The calibration curve was constructed from at least five
non-zero concentrations (at least 14 calibration points).
Calibration points were rejected as described for the IL-2 standard
points. The best-fit regression curve was computed through the
standard points. The R.sup.2 was .gtoreq.0.97. The slope (.beta.)
was .gtoreq.0.77.
Parallelism Analysis
[0137] The dose-response curve of each test sample was plotted on a
log-log scale, with log IL-2 concentration on the y-axis and log GA
DS concentration on the x-axis. The best fit regression curve was
computed through the sample points. The slope (.beta.*) was within
the following range:
.beta..times.0.635.beta.*.beta..times.1.365.
[0138] If .beta.* was out of limits, the in-vitro test was repeated
in duplicate (two separate sample preparations). If .beta.* in one
re-test failed, the batch was rejected. If .beta.* in both re-tests
was within limits, the % potency and 95% fiducial limits were
determined.
Estimation of the % Potency and the Fiducial Limits
[0139] The estimate of the random error to be used to determine the
Fiducial Limits (which have a 95% probability of including the
"true % potency") was obtained by using ANOVA. This statistical
technique splits the total variation between observed responses
into separate components, namely:
TABLE-US-00005 1. due to linear dose-response {close oversize
brace} Model 2. due to the mean effect of preparation 3. due to
deviation from parallelism 4. due to deviation from linearity
{close oversize brace} Random 5. due to residual between-replicate
variation Error
[0140] The components 3 and 4 were included in the random error
term due to non-significant deviations from linearity and
parallelism, respectively. The total sum of squares was partitioned
into 3 components (SS-Regression, SS-Preparation and SS-Error), the
appropriate number of degrees of freedom and the F-test for
significance.
[0141] The % potency of the tested batch was calculated and the 95%
fiducial limits for the estimated potency as described below:
Computational Algorithm for the Calculation of Relative Potency and
95% Fiducial Limits
[0142] Step 1: Compute the transformation of the given data (GA.
Batch and GA. RS.) Into log.sub.10 scale:
[0142] Y.sub.id=log.sub.10(response.sub.id); i=1, . . . n.sub.k
X.sub.id=log.sub.10(dose.sub.id); i=1, . . . n.sub.k
where k=1,2 is a preparation Index of GA, batch and GA, RS,
respectively; n.sub.1 end n.sub.2 are the total numbers of
measurements performed for the GA, batch and GA, RS,
respectively.
[0143] Thus, N=n.sub.1+n.sub.2 is an overall total number of
observations. [0144] Step 2: Calculate the common slope for the
linear regression based on all measured data points vial the
formula:
[0144] .beta. = j = 1 N ( Y j - Y _ ) ( X j - X _ ) j = 1 N ( X j -
X _ ) 2 ; ##EQU00003## [0145] where Y is an overall mean value of
log.sub.10(response); [0146] X is an overall mean value of
log.sub.10(dose). [0147] Step 3: Calculate the sum of squares due
to regression on log.sub.10(dose) as:
[0147] SS REG = .beta. 2 j = 1 N ( X j i - X _ ) 2 ; ##EQU00004##
[0148] Step 4: Calculate the random error sum of squares as:
[0148] SS ERR = k = 1 2 i = 1 n k [ Y ki - Y _ k - .beta. ( X ki -
X _ k ) ] 2 ; ##EQU00005##
where Y.sub.k, X.sub.k are mean log.sub.10(response) and
log.sub.10(dose) values, respectively, of preparation k. [0149]
Step 5: Calculate the Mean Square Error term as following:
[0149] DF.sub.ERR(random error degrees of freedom)=N-3;
MS.sub.ERR=SS.sub.ERR/DF.sub.ERR. [0150] Step 6: Use the
statistical tables of t-distribution in order to find the
appropriate value of t-statistic:
[0150] t=t(0.975,DF.sub.ERR). [0151] Step 7: Calculate the point
estimate of the relative potency as following:
[0151] % Potency = 10 Y _ 1 - Y _ 2 .beta. - ( X _ 1 - X _ 2 ) 100
% ; ##EQU00006## [0152] Step 8: Calculate the expression denoted by
C via the formula:
[0152] C = SS REG SS REG - MS ERR t 2 ; ##EQU00007## [0153] Step 9:
Calculate the logarithms of lower and upper limits of 95% Fiducial
Interval:
[0153] Log 10 ( Lower Limit ) = C Y _ 1 - Y _ 2 .beta. - ( X _ 1 -
X _ 2 ) - MS ERR C t .beta. 1 n 1 + 1 n 2 + ( Y _ 1 - Y _ 2 ) 2 SS
REG - MS ERR t 2 ; ##EQU00008## Log 10 ( Upper Limit ) = C Y _ 1 -
Y _ 2 .beta. - ( X _ 1 - X _ 2 ) + MS ERR C t .beta. 1 n 1 + 1 n 2
+ ( Y _ 1 - Y _ 2 ) 2 SS REG - MS ERR t 2 ; ##EQU00008.2##
Step 10: Transform the values computed in the previous step into
original scale by taking of anti-logarithms of the resulting
log-limits and multiply by 100%.
[0154] The estimated potency of GA DS batch was not less than 80%
and not more than 125% of the stated potency. The fiducial limits
of error (P=0.95) of the estimated potency were less than 70% and
not more than 143% of the stated potency. If the batch was outside
the above limits, the in-vitro test was repeated in duplicate. If
the results of both re-tests were within specifications, the batch
was acceptable. If one re-test failed, the batch was rejected.
Documentation
[0155] The LN cell count and ELISA plates template were recorded.
The original ELISA reader records and the result form were
filed.
Example 2
Development of Standard Procedure of Example 1
[0156] Experiment 2A: Profile of Cytokines Secreted from GA
RS-Specific T Cells
[0157] The LN cells were derived from female (SJL.times.BALB/C)F1
mice immunized with 250 .mu.g GA RS in CFA 9-11 days earlier were
cultured in the presence of various concentrations of GA RS. The
cells were incubated with GA RS for 18-24 hours at 37.degree. C. in
a 5% CO.sub.2 humidified incubator. Subsequently, the cultures were
centrifuged and the supernatants collected and assayed for
cytokines by ELISA.
[0158] The ELISA was performed using biotinylated antibodies
specific to the cytokine and strepavidin-horseradish peroxidase
(HRP) conjugated for detection. Each plate ran included blank
control (first and second antibodies without the cytokine
standard). Each plate ran also included quality control (QC)
samples (three concentrations of cytokine standard within the
assay's linear range). Each in vitro test included a positive
control (Con A, a non-specific T-cell stimulant) and a negative
control (no GA or any other antigen). All the cytokines were
measured after 18-24 hours of incubation. Levels of TGF-.beta.,
IL-10 and IL-4 were tested again after 72 hours of incubation. The
results are shown in Table 4.
TABLE-US-00006 TABLE 4 Cytokine profile Cytokine Secretion levels
IL-2 ++ INF-.gamma. ++ IL-6 + L-10 + L-13 - TGF-.beta. - IL-4 -
TNF-.alpha. -
[0159] In Table 4, the maximal levels measured for each cytokine
are presented in arbitrary units: (-) detection limit; (+) up to
-400 pg/ml; and (++)>400 pg/ml. Table 4 shows that in response
to GA RS in culture, the LN cells secreted IL-2, INF-.gamma., IL-10
and IL-6, while TNF-.alpha., IL-4, IL-13 and TGF-.beta. were not
detected in the culture media. These results indicate that the
cytokines produced by the GA RS-specific T cells are of Th.sub.0
type. It should be noted that a Th.sub.0 profile was observed in
different immunization protocols, i.e., immunization with IFA or
with low doses of GA.
[0160] Since IL-2 is a good marker for T cell activation, and since
the secretion of IL-2 in response to GA RS was very reproducible,
with a linear dose-response relationship, IL-2 seemed to be the
optimum cytokine to measure T cell activation.
Experiment 2B: Optimization of the Immunization Procedure
[0161] Several experiments were performed to establish the optimal
immunization protocol. The first experiment tested the effect of GA
RS (immunizing antigen) dose on T-cell responses in the LN and in
the spleen. Two groups of 10 mice each were immunized with either
250 .mu.g GA in CFA (group 1) (as in the EAE blocking test) or with
10 .mu.g GA in CFA (group 2). Primary cultures were prepared from
both the LN and the spleens of the immunized mice. The cultures
were incubated overnight with various doses of GA RS and afterwards
the culture media were collected and assayed for IL-2 as in
Experiment 2A. The results in FIG. 4 clearly show that in both
immunization protocols the levels of IL-2 secreted from LN cells
are high compared to those secreted from spleen cells. Based on
these results it was decided to use primary cultures of LN cells
for the assay. In addition, the doses of GA RS injected into mice
did not affect the T cell response in culture. Both LN and spleen
cells secreted similar quantities of IL-2 regardless of the
immunizing dose of GA RS. This indicates that the immunization
procedure is robust, and that even major variations in the
immunizing dose of GA RS do not affect the immunological
outcome.
[0162] For further optimization of the immunization protocol, one
group was injected with 250 .mu.g GA RS in CFA and the second group
with 10 mg GA in ICFA. The dose of GA RS in the second group was
higher since ICFA, a weaker adjuvant, was used. Ten days later, the
response of the LN cells from both groups to GA RS was tested in
vitro. FIG. 5 shows that immunization with 250 .mu.g GA RS in CFA
induced a much stronger response in culture, although a much lower
dose of antigen was used.
[0163] Based on these findings, and on the fact that 250
.mu.g/mouse of GA in CFA is very effective in blocking EAE (at
least 80% blocking of EAE in this mouse strain), 250 .mu.g/mouse of
GA in CFA appears to be the optimum dose.
[0164] Specific T cells were usually generated within approximately
10 days, following a single immunization with CFA. FIG. 6 shows the
response of the LN cells to GA RS in culture, prepared 9, 10 and 11
days following immunization. Since the dose-response of IL-2
secretion was similar on all days, the immunization period may last
for 9-11 days.
Experiment 2C: Optimization of the In Vitro Test Conditions
[0165] Several experiments were performed to establish the optimal
protocol for the in-vitro reaction. These studies included
optimization of culture conditions, incubation time, stability of
IL-2 in test samples and stability of GA RS at -20.degree. C.
i) Culture Media
[0166] Cultures of mouse lymphoid cells are usually maintained in
RPMI medium, supplemented with 1% normal mouse serum. Normal mouse
serum may contain endogenous IL-2 that can be detected by the anti
mouse IL-2 monoclonal antibodies used in the ELISA kit. In
addition, the use of different lots of normal serum may increase
the inter-day variations of the in vitro test. To avoid
cross-contamination with endogenous mouse IL-2, and to reduce the
inter-day variations of the method, the responses of the
GA-specific T cells were tested in 4 different culture media: 1)
RPMI+1% normal mouse serum (NMS); 2) RPMI+1% fetal bovine sera
(FBS) (bovine IL-2 is not recognized by the anti mouse IL-2 used in
the ELISA kit); 3) Biotarget (serum-free media produced exclusively
by BeitHaemek, Israel); and 4) DCCM1 (serum-free media produced by
various manufacturers).
[0167] FIG. 7 shows the results of a representative experiment that
compares the response of LN cells to GA RS in different culture
media. The best responses were observed when serum-free media were
used. The dose-response range was left-shifted in the absence of
serum. This can be explained by previous studies showing that GA
binds to albumin and to other serum proteins. This binding may
reduce the availability of GA in culture to interactions with APCs,
and thus higher concentrations of GA are required to stimulate the
T cells. Based on these results, the optimum medium seems to be
DCCM-1 medium.
ii) Kinetics of IL-2 Secretion.
[0168] IL-2 is an autocrine and paracrine growth factor that is
essential for clonal T-cell proliferation and for functional
properties of B cells and macrophages. Following stimulation of the
culture with GA RS, IL-2 is secreted by the activated GA-specific T
cells and is subsequently consumed by the LN cells. Kinetic studies
of IL-2 secretion were performed in an attempt to determine the
optimal (peak) time for collection of the supernatants, following
stimulation with GA. LN cells were cultured and incubated with
various concentrations of GA RS at 37.degree. C. in a humidified
CO.sub.2 incubator. At the intervals indicated in FIG. 8, aliquots
were sampled and the cells were removed by centrifugation. The
supernatants were kept at 20.degree. C. and at the end of the
experiment were assayed for IL-2 by ELISA.
[0169] FIG. 8 shows that the peak of IL-2 levels in the culture
media is between 18-21 hours. The reduction in IL-2 levels in
samples collected from 24-48 hours can be explained by the
consumption of IL-2 by the LN cells. Thus, the optimum time for
supernatant collection appears to be after 18-21 hours of
incubation.
iii) Measurement of Cytokines
[0170] The method relies on accurate measurements of IL-2 in
samples of GA RS and test samples. During the experiments, the
levels of IL-2 were measured by OptEIA (Pharmingen, Cat.
#2614KI)--an ELISA kit specific for mouse IL-2. This ELISA kit is
very sensitive and the results are accurate and reproducible.
iv) Stability of IL-2 in test samples at -20.degree. C.
[0171] In most of the experiments performed, the culture media were
collected and kept at -20.degree. C. before being analyzed by the
ELISA. Preliminary studies of the stability of IL-2 in culture
media show that the cytokine is stable for one week at -20.degree.
C. (FIG. 9). Therefore, the culture media of the in-vitro test
samples can be kept at -20.degree. C. for up to one week prior to
measuring IL-2. The results of this experiment also demonstrated
that the ELISA results are very reproducible--the levels of IL-2
measured in the samples were practically identical in two ELISA
plate runs performed on two different days one week apart.
v) Stability of GA RS Solution at -20.degree. C.
[0172] To test the stability of GA RS solution at -20.degree. C.,
the dose-response of a GA RS solution was tested immediately
following preparation, and after storage for 5 months at
-20.degree. C. FIG. 10 shows that there is practically no
difference in the dose-response curves of GA RS solution before and
after storage for 5 months at -20.degree. C. Therefore, aliquots of
GA RS solution can be prepared and kept at -20.degree. C. for at
least 5 months before use.
Experiment 2D: Determination of Linear Range of GA RS Calibration
Curves
[0173] The statistical validation was carried out based on GA RS
calibration curves calculated and evaluated separately for each one
out of 21 plates received for the analysis. These 21 samples were
gathered at different times over an approximately four-month
period. The GA concentration range for the given plates varied from
0.25 to 50 .mu.g/ml. The following validation characteristics
derived from the GA RS calibration curves constituted the main
concern of the analysis: [0174] 1. Optimal transformation to ensure
wider limits of the linear range; [0175] 2. Determination of the
linear range limits; [0176] 3. Overall criteria for accepting a
calibration curve; [0177] 4. Estimation of assay accuracy and
precision; [0178] 5. Assessment of duplicate reliability (see the
paragraph below).
[0179] The nature of the experiments was such that there were
typically 3 replicates (triplicates) at each calibration point.
However, in some instances, when a triplicate measurement could not
be provided, the assessment of duplicate reliability became
essential.
Linearity of GA Dose-Response Relationship
[0180] The basis of most aspects of the validation discussion
presented below was a linear regression model that related the IL-2
concentration (pg/ml) to the GA concentration (.mu.g/ml). The
assumption of the linearity of this relationship was necessary for
the appropriate fitting of the linear regression model. The data
was plotted in a Linear-Linear scale. The same relationship was
transformed into Log-Log scale, as well as a Log-Linear scale, and
a Log-Square Root scale. The Log-Log transformation demonstrated
the most suitable linear features. Thus, the chosen form of the
regression model was the Log-Log one:
Log.sub.10(IL-2 conc)=a+.beta.*Log.sub.10(GA conc)+error
[0181] The response variable was a log-transformed mean of the 3
replicates measured at each calibration point. This model was
fitted to each calibration sample and the appropriate statistics
(R.sup.2, intercept, and slope) were calculated for each fitted
curve. The value of R.sup.2 reflected the ratio of the residual sum
of squares (RSS) to the total sum of squares (TSS) via the
formula:
R.sup.2=1-RSS/TSS
Linear Range Determination
[0182] The linear range was determined based on the following
criteria: [0183] 1. Visual inspection of plotted log.sub.10 (IL-2
conc) vs. log.sub.10 (GA conc); [0184] 2. The regression influence
diagnostics, such as Cook's known in the art distance statistic;
[0185] 3. Evaluation of the variation of the precision and accuracy
values calculated for the calibration curves for several potential
linear range definitions provided a visual evaluation of the
linearity of the relationship. There was no evidence of
non-linearity of the relationship inside of the chosen linear range
1-25 .mu.g/ml.
Experiment 2E: Determination of Criteria for GA RS Standard
Curve
[0186] Validation parameters derived from GA RS calibration curves,
fitted within selected limits (1-25 .mu.g/ml) of the linear range,
were determined to be the following: [0187] 1. R.sup.2 of the
linear regression fits of log.sub.10 (IL-2 conc) to log.sub.10 (GA
conc) for each plate in the study; [0188] 2. Slopes and intercepts
for these straight line fits; [0189] 3. Accuracy calculated at each
calibration point for each plate; and [0190] 4. Precision
calculated at each calibration point for every plate.
[0191] In order to compute accuracy and precision, each calibration
curve was used to calibrate (back-calculate) the GA Concentrations
given the values of IL-2 concentration:
X.sub.i-back=10.sup.(log.sub.10.sup.(IL-2
Conc).sub.i.sup.-.alpha./.beta.
i=1, 2, 3--triplicate index.
[0192] The basic measure of (in)accuracy used was the percent
difference between the mean of the estimates of concentration and
the true concentration in the triplicate samples:
inaccuracy=([Mean(X.sub.i-back)-GA conc.]/GA conc.)*100%.
[0193] The basic measure of precision used was the relative
standard deviation (RSD or CV) of the triplicate estimates of
concentration:
precision=CV(X.sub.i-back)=[Std. Dev.
(X.sub.i-back)/Mean(X.sub.i-back)]*100%.
[0194] The goal of the analysis was to propose acceptance criteria
for the fitted calibration curve which ensured that the accuracy
and precision of the method were adequate. The acceptance criteria
were based on the R.sup.2 and the slope of the GA RS calibration
curve. About 80% of the plates could be characterized by small
inaccuracy values (<13%) and by good precision (1.1%-6.7%).
[0195] For these 16 "well behaved" standard curves, the following
results were obtained: [0196] 1. High R.sup.2 values (>0.98);
[0197] 2. Relatively high slope values, reflecting dose response
relationships (>0.78 in 15 of 16 plates).
[0198] Since the majority of calibration curves were characterized
by relatively high R.sup.2 (mean=0.99) and by relatively steep
slopes (mean=0.87), in contrast to the excluded plates which had
both relatively low R.sup.2 (mean=0.94) and rather flat slopes
(mean=0.72), the overall acceptance criteria for calibration curves
were considered in terms of R.sup.2 and slope. The simple rule
defining the acceptance parameters was based on the computation of
cut-off points for the slope and R.sup.2 separately and located
them mid-way between the maximum value for rejected curves
(max_R.sup.2=0.95 max_slope=0.77) and the minimum value for
accepted curves (min_R.sup.2=0.98 min_slope=0.77). Thus, the
acceptance criteria were derived as follows: [0199] 1.
R.sup.1.gtoreq.0.97; [0200] 2. Slope.gtoreq.0.77.
[0201] These criteria were applied to at least five different
(triplicate) concentrations for fitting the calibration curve
within the range 1-25 .mu.g/ml of GA concentration. Additionally,
the range of intercept values was between 1.42-1.78, mean=1.58.
This range was similar for the 16 eligible and the 5 removed
plates.
[0202] Accuracy and precision were calculated for each curve, and
for each concentration among those on the plate. These individual
values (for each curve and concentration) were also averaged over:
[0203] 1. Different concentrations for each curve; [0204] 2.
Different curves for each concentration; and [0205] 3. Over all
curves and concentrations.
[0206] The relevant conclusion was that for GA RS calibration
curves based on at least five different calibration points in the
linear range 1-25 .mu.g/ml, when the calibration curve was
restricted to having R.sup.2.gtoreq.0.97 and slope.gtoreq.0.77, the
resultant average accuracy and precision was estimated as: [0207]
1. The mean (.+-.SD) accuracy value for the method was:
8.0%.+-.2.3%; and [0208] 2. The mean (.+-.SD) precision value for
the method was: 2.9%.+-.1.7%.
Reliability Assessment of GA RS Calibration Curves Based on
Duplicate Measurements
[0209] A comparison of the assay's accuracy and precision
descriptive statistics was performed in order to assess the
reliability of GA RS calibration curves fitted using duplicate
measurements at each calibration point. In addition, the individual
accuracy and precision values (for each curve and concentration)
for all three possible selections of duplicate measurements, out of
the given triplicate, were studied. When concentrating on those
curves that satisfied the acceptance criteria described in the
previous section and fitted within the limits of the defined linear
range 1-25 .mu.g/ml, it was evident that when a triplicate
measurement can not be provided for some reasons, it can be
successfully substituted by duplicate measurement.
TABLE-US-00007 TABLE 5 Accuracy and Precision Descriptive
Statistics Summary Triplicate Duplicate (1, 2) Duplicate (1, 3)
Duplicate (2, 3) Accuracy Precision Accuracy Precision Accuracy
Precision Accuracy Precision Mean 8.00 2.93 8.13 2.53 7.98 2.88
8.40 2.40 S.D. 2.31 1.72 2.80 1.66 2.45 2.13 2.79 1.53 Min 5.23
1.10 4.93 0.95 5.21 0.93 4.97 0.81 Max 12.79 6.67 12.51 6.33 12.44
9.24 13.41 5.99
[0210] The mean (.+-.SD) accuracy and precision of the method based
on triplicates were 8.0%.+-.2.3% and 2.9%.+-.1.7%, respectively
(Table 5).
[0211] The mean (.+-.SD) accuracy and precision of the method based
on duplicates were: [0212] 1. Accuracy: 8.1%.+-.2.8%; Precision:
2.5%.+-.1.7%; [0213] 2. Accuracy: 8.0%.+-.2.5%; Precision:
2.9%.+-.2.1%; and [0214] 3. Accuracy: 8.4%.+-.2.8%; Precision:
2.4%.+-.1.5%.
Experiment 2F: Determination of Statistical Relationship
[0215] It was found that the mean (in)accuracy of the method is
8.0% with SD=2.3%. The aim was to develop a reliable test for the
slope comparison of two log(dose)-log(response) lines of a new GA
batch vs. GA RS. The test took into account the (in)accuracy of the
above-mentioned method. The highest limit of the approximate 95%
individual tolerance region for the mean (in)accuracy of the method
served as a threshold value: Mean+2*SD=12.6%. Thus, variations
within the range.+-.12.6% were considered non-significant.
[0216] A full mathematical explanation of the relationship between
.beta.* (the slope of the batch line), .beta. (the slope of the
standard line) and the highest permitted (in)accuracy value
follows. Without loss of generality, only the case where
.beta.*>.beta. will be proved in detail (due to the existing
symmetry, the extension of the proof for the case where
.beta.*<.beta. is obvious). The back-calculated dose value, for
a given log(response) was:
X.sub.back=10.sup.(Y-.alpha.)/.beta. where Y=log.sub.10 (IL-2
concentration).
The formula for the (in)accuracy calculation was:
(in)accuracy=[(10.sup.(Y-.alpha.)/.beta.-X.sub.true)/X.sub.true]*100%.
Y.sub.low and Y.sub.high were the lowest and highest log(response)
values permitted by the highest allowable (in)accuracy of
.+-.12.6%.
[0217] Thus, the region where the hypothesis of the equality of
slopes was to be accepted was:
{ [ ( 10 ( Y low - .alpha. ) / .beta. X 1 ) / X 1 ] * 100 %
.gtoreq. - 12.6 % [ ( 10 ( Y high - .alpha. ) / .beta. X 2 ) / X 2
] * 100 % .ltoreq. - 12.6 % { 10 ( Y low - .alpha. ) / .beta. / X 1
.gtoreq. 0.874 10 ( Y high - .alpha. ) / .beta. / X 2 .ltoreq.
1.126 ##EQU00009##
[0218] Thus, the boundaries of the equality of the slopes were:
{ Y low = .alpha. + .beta. log ( X 1 0.874 ) Y high = .alpha. +
.beta. log ( X 2 1.126 ) ##EQU00010##
[0219] The slope of a straight line was calculated as follows:
.beta.*=(Y.sub.high-Y.sub.low)/(log X.sub.2-log
X.sub.1)=[.beta.-log([X.sub.2/X.sub.1][1.126/08.74])]/log(X.sub.2/X.sub.1-
)
.beta.=[.beta.log(1.288)]/log(X.sub.2/X.sub.1)=.beta.(1+log(1.288)/log(X-
.sub.2/X.sub.1))
[0220] Assuming for the particular case under consideration that
X.sub.2/X.sub.1=2 (for dose levels of 5 and 10 .mu.g/ml), .beta.*
was calculated as follows:
.beta.*=.beta.(1+log(1.288)/log 2)=.beta.1.365
[0221] Combining this result with the one obtained for the
symmetric case where .beta.*<.beta., the limits were calculated
as:
{ .beta. * .ltoreq. .beta. 1.365 .beta. * .gtoreq. .beta. 0.635
##EQU00011##
[0222] In the given data, all slope values were within the matching
critical limits, meaning that no deviation from the parallelism
assumption was observed.
[0223] Once a batch was accepted as statistically valid (existence
of linearity and parallelism has been proved), the potency ratio of
the test preparation relative to the standard was estimated. This
was done in a parallel line assay by fitting straight parallel
lines to the data and determining the horizontal distance between
them:
M = log .rho. = Y _ T - Y _ S B - ( X _ T - X _ S ) ;
##EQU00012##
where .rho.denoted the potency, Y.sub.S, Y.sub.T, X.sub.S, X.sub.T,
were the mean log(responses) and log(doses) of the standard and
test preparations, respectively. B--was a common slope for the
standard and test log(dose)-log(response) lines. The least-squares
estimate of the common slope--B was a weighted average of the
least-squares estimates of two slopes separately from the standard
line and the test line. Taking the anti-logarithm of the expression
above, one was able to obtain a point estimate of the "true %
potency" of a test preparation relatively to its standard:
% Potency = 10 Y _ T - Y _ S .beta. - ( X _ T - X _ S ) 100 % .
##EQU00013##
Example 3
Validation of the Standard Procedure of Example 1
[0224] The goal of the analysis, presented below, was to establish
validated release specifications for the relative potency of a GA
batch. A GA batch was considered valid, if the following criteria,
based on statistical inference, were fulfilled: [0225] 1. No
violations of the assumptions involved in the bioassay analysis
approach: [0226] (a) Independence and normality of the
log(responses); [0227] (b) Homogeneity of the variance of the
log(responses); [0228] (c) No outliers; [0229] (d) Parallelism
(non-significance of the slope ratio test); [0230] 2. The point
estimate of the relative potency was within a pre-specified range:
80%-125%; and [0231] 3. The 95% Fiducial Limits for the "true
relative potency" value were within a wider pre-defined confidence
range: 70% to 143%.
[0232] The model assumed that the standard and the test
preparations should behave as if one were a simple dilution of the
other. This means that the log(dose)-response lines for the two
preparations should not deviate significantly from linearity and
parallelism. Thus, an anti-logarithm of the constant horizontal
displacement between these straight lines was able to serve as an
estimate of the potency ratio. These two requirements, linearity
and parallelism, constituted a concept of the assay validity. The
check of validity was a prerequiste to the estimation of the
relative potency and its fiducial limits.
[0233] The estimate of random error was needed for the computation
of fiducial limits for the true value of the relative potency. This
measure was obtained by the implementation of the statistical
technique known as "Analysis of Variance" (ANOVA). Therefore, the
classical statistical assumptions of the ANOVA must have been
satisfied. The requirements for the statistical analysis of a
parallel-line bioassay model were as follows: [0234] 1. The
responses were independently normally distributed about their
expected values; [0235] 2. The variance of the response was not
affected by the mean response value; [0236] 3. There were no
outliers; [0237] 4. The relationship between the log(dose) and
response was able to represented by a straight line over the range
of doses; and [0238] 5. The straight line of the test preparation
was parallel to that of the standard.
[0239] The batch analysis data was obtained from different
experiments performed on different days by different operators.
Validation trials of the standard procedure of Example 1 were
carried out by a series of experiments, each involving: 1)
immunization of mice with 250 .mu.g GA RS in CFA; 2) preparation of
a primary culture from the LN cells 9-11 days following
immunization; 3) incubation of the LN cells with various
concentrations of GA RS and with test samples; 4) collection of the
culture media and analysis of IL-2 levels by ELISA; 5) plotting a
GA RS curve based on triplicate IL-2 measurements performed at 6
dose levels from 1-25 .mu.g/ml; and 6) comparison of the T cell
response to each test sample to the response to the RS batch (in
triplicate) at two concentrations within the linear range (5 and 10
.mu.g/ml). The % CV was also calculated for each triplicate in
order to detect any problems associated with variability between
triplicates (normally, the % CV between triplicates should not
exceed 10-15%). For the given data, no violations of the conditions
were detected.
[0240] The validation characteristics used to provide an overall
knowledge of the capabilities of the analytical procedure were:
linearity, range, accuracy, precision, specificity and robustness.
The validation criteria and analyses were based on the ICH
consensus guideline, "Validation of Analytical Procedures:
Methodology", November 1996 (CPMP/ICH/281/95).
[0241] Statistical methods recommended in "European Pharmacopoeia"
guideline were adapted to the given data for analysis purposes.
Experiment 3A: Linearity and Range
[0242] In each in vitro test, a dose-response curve of GA RS batch
was used to calculate the relative response of the cells to the
tested samples. Each calibration curve included at least five
points (without zero). Twenty-one calibration curves collected from
different in vitro tests, performed during the development and the
validation stages, were plotted and evaluated for each plate.
[0243] Statistical analysis of the data revealed that the plots of
log.sub.10 (IL-2 concentration) versus log.sub.10 (GA RS
concentration) provided the best linear fit. The linear range
mainly emerged by visual inspection and evaluation of accuracy and
precision of the calibration points. The % RSD was calculated for
each triplicate in order to detect any problems associated with
variability between triplicates (normally, the % RSD between
triplicates should not exceed 10-15%). The range of GA RS curve was
specified between 1-25 .mu.g/ml.
[0244] Based on these analyses, the GA RS curve should be comprised
of at least 6 calibration points, one with zero concentration
(negative control) and at least 5 concentrations of GA RS in the
range between 1 and 25 .mu.g/ml. Linear regression of log.sub.10
(IL-2 concentration) versus log.sub.10 (GA RS concentration) should
have an R.sup.2.gtoreq.0.97 and a slope.gtoreq.0.77.
Experiment 3B: Accuracy
[0245] The accuracy of the method was established across the
specified linear range of the GA RS curve. Statistical analysis of
the data revealed that the mean accuracy of the method was:
8.0%.+-.2.3%.
Experiment 3C: Precision
[0246] The basic measure of precision used was the relative
standard deviation (RSD) of replicate (usually triplicate) estimate
of concentration.
[0247] The RSD was established across the linear range of GA RS
curves. Statistical analyses of the data revealed that the mean
precision of the method was 2.9%.+-.1.7%. The reliability of
duplicate measures was equivalent to that of triplicate. Therefore,
when one of the three replicates was identified as an outlier, the
outlier was omitted and the results from duplicate measures were
accepted.
Experiment 3D: Method Repeatability
[0248] The GA specific T cell response to a GA DS batch was
measured repeatedly, 3 times, in the same in vitro test. Three
weights of the same batch were each diluted to 5 and 10 .mu.g/ml
and incubated with the GA-specific T-cells. The levels of IL-2 in
the culture media of the test samples and of the GA RS samples,
were measured by ELISA in triplicate. The % potency and 95%
fiducial limits of the cells to each replicate were calculated
relative to the GA RS. Table 6 shows the % response calculated for
each replicate.
TABLE-US-00008 TABLE 6 Method Repeatability 95% Fiducial GA DS
Limits (Lower Sample conc. AVG Limit-Upper # (.mu.g/ml) % Potency N
= 6 SD RSD Limit) 1 5 75 80 74 77 3.3 4.2 67-89 10 73 79 81 2 5 83
83 92 84 5.0 5.9 73-97 10 80 79 88 3 5 72 74 71 76 5.2 5.2 65-88 10
80 78 79
Experiment 3E: Intermediate Precision
[0249] The % response of a GA DS batch was tested in 3 different in
vitro teats, performed in different days, by 3 different
investigators from the same laboratory. Table 7 summarizes the %
potency and 95% fiducial limits determined for this batch in the 3
repeated experiments.
TABLE-US-00009 TABLE 7 Intermediate Precision 95% Fiducial GA DS
Limits (Lower Test conc. AVG Limit-Upper # (.mu.g/ml) % Potency N =
6 SD RSD Limit) 1 5 85 86 83 83 3.1 3.7 71-97 10 77 84 84 2 5 90 86
87 86 3.0 3.7 79-94 10 82 84 89 3 5 75 80 74 77 3.3 4.2 65-88 10 73
79 81
Experiment 3F: Method Reproducibility
[0250] The reproducibility of the method was assessed by means of
inter-laboratory study. The % response GA DS batch was tested in
two different experiments, performed in 2 different laboratories,
using different analysts, equipment and reagents. Table 8
summarizes the results from both labs.
TABLE-US-00010 TABLE 8 Method Reproducibility Lab 1 Lab 2 Avg %
Potency .+-. RSD 86 .+-. 3.5 83 .+-. 5.0 %95 Fiducial Limits 79-94
78-90
[0251] Based on the above experiments, it can be concluded that the
in vitro test is reproducible.
Experiment 3G: Specificity
[0252] The discrimination of the method was tested at 3 levels: 1)
discrimination between samples incubated with/without GA RS (matrix
effect); 2) discrimination between GA RS and other related and
non-related proteins and peptides, including GA DS; and 3)
discrimination between GA RS and GA related copolymers in which the
peptide sequences have been deliberately modified.
i) Recognition of GA RS by GA RS-Specific T Cells (Matrix
Effect)
[0253] GA-specific T cells were induced by immunization of female
(SJL.times.BALB/C)F1 mice mouse with 250 g GA RS in CFA. This dose
of GA is routinely used for testing the biological activity of GA
batches in this mouse strain using the EAE blocking test. The
control group in this experiment was injected with CFA alone. Ten
days following immunization, LN cells were removed from the both
groups of mice. The cells were incubated with GA RS for 18-24 hours
at 37.degree. C. in a 5% CO.sub.2 humidified incubator.
[0254] Subsequently, the cultures were centrifuged and the
supernatants collected and assayed for interleukin-2 (IL-2, a
cytokine secreted from activated T cells) by ELISA using
biotinylated antibodies specific to IL-2 and
strepavidin-horseradish peroxidase (HRP) conjugate for detection
(FIG. 1). Each plate run included blank control (first and second
antibodies without the cytokine standard). Each plate run also
included quality control (QC) samples (three concentrations of
cytokine standard within the assay's linear range). Each in vitro
test included a positive control (Con A, a non-specific T-cell
stimulant) and a negative control (no GA or any other antigen).
FIG. 2 shows that the LN cells from mice immunized with GA RS
secrete IL-2 dose dependently in response to GA RS in culture,
while LN cells from the control mice do not respond to GA. RS in
culture. The levels of IL-2 in the negative control samples is
usually below or close to the ELISA detection limit (approximately
3 .mu.g/ml). These IL-2 levels are always below the levels secreted
by the lowest calibration point of GA RS (1 .mu.g/ml). These
results indicate that the secretion of IL-2 by the GA specific
T-cells is GA dependent.
ii) Discrimination between related and non-related antigens
[0255] The discrimination between related and non-related antigens
(proteins and single peptides) was demonstrated by testing the
response of the GA RS-specific T cells to various antigens
in-vitro. A primary culture of LN cells derived from female
(SJL.times.BALB/C)F1 mice immunized 9-11 days earlier with 250
.mu.g GA RS in CFA. The primary culture was incubated overnight
with GA RS and with various other antigens at 37.degree. C. in a 5%
CO.sub.2 humidified incubator. Then, the cultures were centrifuged
and the supernatants collected and assayed for IL-2 by ELISA as in
Experiment 3G(i).
[0256] Table 9 shows that in this experimental system the
GA-specific T cells did not respond to either human MBP (myelin
basic protein), the MBP immunodominant peptide pp. 87-99 (an
encephalitogenic peptide), or its analog pp. 87-99.sub.Ala 76 (an
EAE suppressor peptide). Lysozyme, a non-relevant basic protein,
was also not recognized by the GA-specific T cells. TV-35 and
TV-109 were peptides with a molecular weight of 3757 and 11727,
respectively (PCT International Publication No. WO 00/18794). These
peptides had a defined sequence comprised from the same four amino
acids of GA (Ala, Glu, Lys, Tyr), in the same molar ratio as in GA.
The GA RS-specific LN cells did not respond to TV-35, and had a
very low cross-reactivity with TV-109. These results can be
explained by the observation that immunization with GA RS induced
the formation of a mixture of T cells with different specificity
towards the multiple T-cell epitopes present in GA. TV-35 and
TV-109 may share common sequences with GA, however, and incubation
of the GA-specific T cells with a single peptide probably caused
only a partial stimulation of a small fraction of the GA-specific
cells in culture. Thus, the overall T-cell response (secretion of
IL-2) was below or close to detection limits.
TABLE-US-00011 TABLE 9 Specificity of GA RS-specific LN cells
Antigen % Potency.sup.1 GA RS 100 Lysozyme 0 Human MBP 0 MBP pp.
87-99 0 MBP pp. 87-99.sub.Ala 96 0 TV-35 0 TV-109 17 1 % Potency =
IL - 2 concentration in culture media of the antigen .times. 100 IL
- 2 concentration in culture media of GA RS ##EQU00014##
[0257] The in vitro test was sensitive to the average molecular
weight (MW) of the GA batch. FIG. 11 shows the response of the GA
RS-specific cells to GA RS (MW=7900) and to GA DS batches differing
in their average MW. As can be seen, the response generally
correlated with the average MW; the higher the average MW, the
greater the response. However, it should be noted that the release
specifications for the average MW of GA DS are between 4700-10000,
and that similar levels of IL-2 were secreted in response to DS
batches with average MW within specifications (FIG. 11). These
results indicate that the method was highly specific to GA and
sensitive to changes in the average MW of GA.
iii) Recognition of GA drug substance (DS) and Copaxone.RTM. Drug
Product (DP) by GA RS-Specific T Cells
[0258] Nine to eleven days following immunization of female
(SJL.times.BALB/C)F1 mice with 250 .mu.g GA RS in CFA, the LN cells
were removed and cultured with various doses of GA RS batch (the
immunizing antigen) and with a DS batch.
[0259] IL-2 was measured as in Experiment 3G(i). FIG. 12A shows
that the LN cells cross-reacted with both standard batches. The
dose-response curves of IL-2 secretion (measured by ELISA as above)
by both batches were similar, indicating that the tested batches
shared similar T-cell epitopes. Comparison between GA RS and a
Copaxone.RTM. batch shows that the GA RS-specific T cells also
cross-reacted with the DP batch, and that mannitol, the excipient
in the Copaxone.RTM. formulation, did not affect or interfere with
the T-cell responses FIG. 12B). Thus, this method provides an
indication of batch-to-batch reproducibility.
v) Discrimination Between GA and Related Copolymers
[0260] In Experiment 3G(ii), it was demonstrated that the in vitro
test was, sensitive to the average MW of GA peptides, using GA DS
batches differing in their average MW. Since the experiment was
based on bio-recognition of GA by GA-specific T cells, which
specifically respond to linear sequences, it was expected that the
method would be sensitive to variations/modifications in the
sequences of GA peptides. This was demonstrated by using: 1)
copolymers synthesized from only 3 out of the 4 amino acids
comprising GA; 2) a GA batch (XX) resulting from deliberate
modification in manufacturing conditions, i.e., addition of excess
of free amino acids to GA monomers during synthesis. The average MW
of this batch was high and out of specifications (MW=11150 Da); and
3) degradation products of GA RS obtained by proteolysis with
trypsin and chymotrypsin.
[0261] Table 10 shows that the GA-specific T-cells did not respond
to the 3 amino acid copolymers lacking lysine, alanine or tyrosine.
In addition, the % response of the cells to the batch XX was
relatively high and out of the method specifications (100.+-.30%),
indicating that the method might be sensitive to modifications in
the production process. The high % response can also be explained
by the sensitivity of the test to the MW of GA peptides, as
demonstrated.
TABLE-US-00012 TABLE 10 Method Specificity Copolymer Modification
Average % Potency .+-. RSD Tyr-Lys-Glu Lacking Alanine 0
Tyr-Ala-Glu Lacking Lysine 0 Ala-Glu-Lys Lacking Tyrosine 0 Batch
XX Excess of free amino 170 .+-. 4.7 acids in polymerization
stage
[0262] Kinetics studies of GA RS proteolysis by trypsin and
chymotrypsin show that the in vitro test was sensitive to
degradation of GA peptides. FIGS. 13 and 15 show that the secretion
of IL-2 by the cells was reduced upon proteolysis time, and the %
potency of the cells to the proteolysed peptides was out of the
method specifications (100.+-.30%) (Table 11). Overlay
chromatograms (by RP-HPLC) of the degraded samples (FIGS. 14 and
16) demonstrated the kinetics of the proteolysis by trypsin and
chymotrypsin, respectively. The cumulative results from all
specificity studies revealed that the method was highly specific to
GA and discriminated between GA and closely related antigens.
TABLE-US-00013 TABLE 11 Method Specificity - Effect of Proteolysis
Time of proteolysis Enzyme (minutes) Average % Potency Trypsin 1 40
5 36 15 19 30 7 overnight 0 Chymotrypsin 5 11 20 0
Experiment 3G: Robustness
i) Robustness of Acceptance Criteria
[0263] The consistency and robustness of the defined acceptance
criteria was examined by comparing the resulting estimates of the
relative potency obtained for the repeated GA batches. The batch
analysis data included a number of repeated GA batches. Two batches
were measured on three different days by different operators. One
batch was tested on two different days by different operators, as
well.
[0264] In the parallelism test for the repeated GA batches, all GA
batch slopes values were within the appropriate critical limits for
the parallelism slope ratio test. All GA batches satisfied the
acceptance criteria for the point estimates of the relative potency
values with 95% fiducial limits (the estimated % potency was within
the limits of 80%-125% and the 95% fiducial limits were within the
range of 70%-143%). For the analyzed data of the repeated GA
batches, their validity did not depend on the day of experiment or
the operator performing the test. This data supports the robustness
of established specifications.
ii) Robustness of Critical Parameters in the Immunization Procedure
and the In Vitro Reaction
[0265] The robustness of critical parameters in both the
immunization procedure and the in vitro reaction was evaluated.
Briefly, it was shown that: 1) the immunological response of the LN
cells was not affected by the immunizing dose of GA RS; 2) the
immunization period was 9-11 days; 3) the response of the LN cells
to GA RS was higher compared to the spleen cells response; 4)
immunization with GA RS+CFA resulted in the LN cells having a
stronger response compared to immunization with ICFA; 5) the
presence of serum in culture media strongly affected the
GA-specific T cell response, thus the in vitro reaction was
performed in a serum-free media; 6) the optimal time frame for
collecting the culture media was 18-21 hours following incubation
with GA RS and test samples; and 7) the culture media can be kept
at -20.degree. C. for up to one week before tested in ELISA. Thus,
it was shown that the method was robust.
Summary Statistics for the Point Estimate and 95% Fiducial Limits
of Relative Potency
95% Tolerance Limits for the Mean Relative Potency
[0266] To assess the acceptance limits for the estimated relative
potency of a new batch, the mean and the standard deviation of the
individual log(potency) estimates were calculated:
Mean(M.sub.i)=0.0074;
SD(M.sub.i)=0.0402.
[0267] An approximated 95% tolerance range for the mean relative
potency value, based on the analyzed data, was:
[10.sup.Mean(M.sup.i.sup..+-.2*SD(M.sup.i.sup.)]*100%=[84%,122%].
Range of the 95% Fiducial Limits of Relative Potency
[0268] The minimum and maximum values of the 95% Fiducial Limits
for the individual relative potency estimates were: [0269]
Minimum(Low Limit)=79.3% [0270] Maximum(High Limit)=147.3%
Satisfaction of the Acceptance Criteria
[0271] Based on the analysis, the acceptance criteria were
determined to be: [0272] 1. The assumptions involved in bioassay
analysis approach were fulfilled, namely: [0273] a. Independence
and normality of the log(responses); [0274] b. Homogeneity of the
variance of the log(responses); [0275] c. No outliers; and [0276]
d. Parallelism (non-significance of the slope ratio test). [0277]
2. The estimated relative potency was not less than 80% and not
more than 125% of the standard potency; and [0278] 3. The 95%
Fiducial Limits of error of the estimated relative potency were not
less than 70% and not more than 143% of the standard potency.
Discussion of Example 3
[0279] Validation of the in vitro test revealed that the method was
reproducible and the mean accuracy and precision were in an
acceptable range. The method was highly specific to GA peptides and
sensitive to the quality of the active substance.
Summary and Discussion
[0280] An in vitro method was developed for GA DS and Copaxone.RTM.
batches. This method was based on bio-recognition of T-cell
epitopes (linear sequences) by GA RS-specific T cells. The GA
RS-specific T cells secrete Th.sub.0 cytokines in response to GA in
culture. In this method, the recognition of GA batches by T cells
is monitored by measuring the levels of IL-2 in the culture media
by ELISA. It was shown that the GA RS-specific T cells are
cross-reactive with both DS and DP batches, indicating that these
batches share similar sequences with the RS batch, and that
mannitol, the excipient in the DP formulation, does not interfere
with the reaction.
[0281] The method was very specific to GA peptides and is sensitive
to the average MW of the peptide mixture. MBP was not recognized by
the GA-specific T cells. MBP immunodominant peptides (both
encephalitogenic and suppressive peptides), as well as single
peptides with amino-acid composition similar to that of GA, did not
stimulate the T cells. Critical parameters in the immunization
procedure, as well as in the in-vitro reaction, were optimized
during this experiment. This experiment showed that the method was
very reproducible and robust.
[0282] The method can be adapted to standardize other T cell
antigens for use in pharmaceutical compositions. A primary culture
of T cells specific to an antigen RS, instead of GA RS, can be made
from animals immunized against the antigen RS. The cytokine
production of this culture in response to antigen RS and in
response to the sample antigen can be measured. The cytokine
production in response to antigen RS can be plotted against the
concentration of antigen RS to create a standard curve. The
cytokine production in response to the sample antigen can be
compared to the standard curve to determine whether the antigen is
within the acceptable range of potency.
[0283] The optimum cytokine to monitor can be determined as in
Experiment 2A. Conditions for immunization and the in vitro test
may be optimized as in Experiments 2B and C.
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