CHARACTERIZING A GLAT RAMER ACETATE RELATED DRUG PRODUCT
This application claims priority of U.S. Provisional Application
Nos. 61/819,481, filed May 3 , 2013 and 61/749,228, filed January 4 ,
2013, the contents of each of which are hereby incorporated by
reference in their entireties.
Throughout this application various publications are referenced by
numerical identifiers in parentheses. Full citations of these
references can be found following the Examples. The disclosures of
these publications in their entireties are hereby incorporated by
reference into this application in order to more fully describe the
state of the art to which this invention pertains.
Multiple sclerosis (MS) is a chronic, debilitating autoimmune
disease of the central nervous system (CNS) with either relapsingremitting
(RR) or progressive course leading to neurologic
deterioration and disability. At time of initial diagnosis, RRMS is
the most common form of the disease (1) which is characterized by
unpredictable acute episodes of neurological dysfunction (relapses) ,
followed by variable recovery and periods of clinical stability. The
vast majority of RRMS patients eventually develop secondary
progressive (SP) disease with or without superimposed relapses.
Around 15% of patients develop a sustained deterioration of their
neurological function from the beginning; this form is called
primary progressive (PP) MS. Patients who have experienced a single
clinical event (Clinically Isolated Syndrome or "CIS") and who show
lesion dissemination on subsequent magnetic resonance imaging (MRI)
scans according to McDonald's criteria, are also considered as
having relapsing MS. (2)
With a prevalence that varies considerably around the world, MS is
the most common cause of chronic neurological disability in young
adults. (3, 4 ) Anderson et al . estimated that there were about
350,000 physician-diagnosed patients with MS in the United States in
1990 (approx. 140 per 100,000 population) .(5) It is estimated that
about 2.5 million individuals are affected worldwide. (6) In general,
there has been a trend toward an increasing prevalence and incidence
of MS worldwide, but the reasons for this trend are not fully
understood. (5)
Current therapeutic approaches consist of i ) symptomatic treatment
ii) treatment of acute relapses with corticosteroids and iii)
treatment aimed to modify the course of the disease. Currently
approved therapies target the inflammatory processes of the disease.
Most of them are considered to act as immunomodulators but their
mechanisms of action have not been completely elucidated.
Immunosuppressants or cytotoxic agents are also used in some
patients after failure of conventional therapies. Several
medications have been approved and clinically ascertained as
efficacious for the treatment of RR-MS ; including BETASERON®,
AVONEX® and REBIF®, which are derivatives of the cytokine interferon
beta (IFNB) , whose mechanism of action in MS is generally attributed
to its immunomodulatory effects, antagonizing pro- inflammatory
reactions and inducing suppressor cells. (7) Other approved drugs for
the treatment of MS include Mitoxantrone and Natalizumab.
Copaxone ® (Teva Pharmaceutical Industries Ltd.) is a glatiramer
acetate drug product approved for treatment of patients with
relapsing-remitting multiple sclerosis (RRMS) and clinically
isolated syndrome (CIS) (8) . Glatiramer acetate drug substance (GA) ,
the active substance of Copaxone ® , is a complex mixture of
polypeptides and is the first member of the glatiramoid class; i.e.,
a complex mixture of synthetic polypeptides of varying sizes
assembled from four naturally occurring amino acids: L-glutamic
acid, L-alanine, L-lysine, and L-tyrosine, in a defined molar ratio
(9).
GA elicits an i-inflammatory as well as neuroprotective effects in
various animal models of chronic inflammatory and neurodegenerative
diseases (10-14) and has been shown to be safe and effective in
reducing relapses and delaying neurologic disability in MS patients
following long-term treatment (15).
The mechanisms underlying GA therapeutic activity are not fully
elucidated, but GA activity on immune cells has been well
demonstrated. GA appears to act as an altered peptide ligand (APL)
of encephalitogenic epitopes within myelin basic protein (MBP) (16)
and demonstrates cross-reactivity with MBP at the humoral and
cellular levels (17-23) . The unique antigenic sequences of the GA
polypeptide mixture compete with myelin antigens for binding to MHC
class II molecules on antigen presenting cells (APCs) and
presentation to the T cell receptor (TCR) , resulting in the
induction of anergy or deletion of autoreactive MBP-reactive T cells
and proliferation of GA-reactive T cells. At initiation of Copaxone
treatment, GA-reactive CD4+ T-cell lines from MS patients secrete
both pro- inflammatory T helper type 1 (Thl) and anti-inflammatory
Th2 cytokines (21, 24) , but continued exposure to Copaxone induces a
shift in GA-reactive T cells toward the Th2 phenotype (21, 23, 25-
28).
Copaxone also increases the number and suppressive capacity of
CD4+CD25+FOXP3+ regulatory T cells, which are functionally impaired
in MS patients (29-31) . Furthermore, treatment leads to antigennonspecific
modulation of APC function. Copaxone treatment promotes
development of anti- inflammatory type II monocytes characterized by
an increase in interleukin (IL)-IO and transforming growth factorbeta
(TGF- b) and decreased production of IL-12 and tumor necrosis
factor (TNF) (32) .
S & Y OF THE INVENTION
High- throughput gene expression analysis was used to further
characterize the functional pathways that are modulated by GA within
immune cells. This technique facilitates investigation of thousands
of genes and allows identification of a wide range of biological
functions. Microarray gene expression analyses were conducted using
GA-primed murine splenocytes reactivated ex vivo with GA or with a
variant referred to as GA-Natco (Glatimer®, Natco Pharma, Ltd. ,
Hyderabad, India) . The transcriptional alteration of genes induced by
GA or GA-Natco, were evaluated with respect to functional pathways
that may be related to known mechanisms of GA activity. This
sensitive high- throughput gene expression analysis sheds some light
on the mode of action of GA and on differences between various
glatiramoids that are otherwise difficult to detect.
The present invention provides a process for characterizing a
glatiramer acetate related drug substance or drug product comprising
the steps of:
a ) obtaining a batch of the glatiramer acetate related drug
substance or drug product;
b ) immunizing a mammal with a predetermined amount of a
glatiramer acetate related drug substance or drug product;
c ) preparing a culture of cells from the mammal of step b ) at a
predetermined time after immunization;
d ) incubating cells from the culture of step c ) with a
predetermined amount of the glatiramer acetate drug related
substance or drug product of step a ) ; and
e ) determining the level of expression of at least one gene
selected from the group consisting of genes regulated by
glatiramer acetate reference standard or glatiramer acetate
drug substance or drug product in Gene Expression Omnibus
accession number GSE40566; determining the level of expression
of at least one gene selected from the group consisting of
Ecml, Presl, Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590,
Gprll4, Tmie, Rasgrpl, Myo6 , Pfkp, Uspl8, Arl4c, Als2cl,
2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil,
4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igfbp4, Oas2,
Bclllb, Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl, Chi311, Anxa3 ,
Hp, Lyz2, Lyz, Ferll3, Sirpa, Cd63, Clec4n, Clec4d, EG433016,
Stfal, Chi313 Ngp, S100a8, S100a9, Clecsf9, Saa3 ,
5033414K04Rik, Slc7all, Slpi, Cdl4, Fpr2, Fcgr3 , F10, Gpnmb,
Tgfbi, Mmpl4, Slcllal, C3 , Gpr84, Acta2, Lcn2 , Hmoxl, Tpsabl,
Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb,
Cxcl2, Ilia, Ccl3, 6720418B0lRik, 583 0496LllRik, Cd8bl , Fcgrt,
LOC385615 and Scml4; determining the level of expression of at
least one gene selected from the group consisting of CD40,
CD86, GATA3, HLA-DMA, HLA-DMB, ICOS, IFNG, IFNGR2 , IL2, IL13 ,
IL4, IL18, IL12RB1, IL17A, IL17F, IL18R1, IL2RA, IL2RG, IL4R,
IL6R, TBX21, TGFBR2 , TNF, FOXP3 , IL10RB, KLRD1, CD69, LTB,
CD83, PRFl, CAMK2D, LTA, FSCNl , TLR7 , CSF2 , CCR7 , FASLG, ILIA,
CCL5, CD8B, CXCL10, TLR2 , CCL4, TLR7 , IGHAl, IL24, SOCS1,
OAS1, JAK1, PTPN2, IFITMl, IFI35, S A 2 , BCL2 , MVD, FDPS,
SQLE, NSDHL, DHCR24, Acat2/Acat3 , MSMOl, LSS, CYP51A1, NFKBIE,
PIK3R1, PPP3CC, CD3D, IL2RB, PTEN, CD3G, ICOS , CAMK2D , NFAT5 ,
LAT, ITK, H2-M2, FASLG, LIF, IGHAl, PRKACB, SGKl , MAPK11,
TSC22D3 , JUN, FKBP5 , ADRB2 , MAP3K1, MAPK12, POU2F1, SMARCA2 ,
CDKN1A, TGFB3, HSP90AA1, DHCR24, CCR5 , and CXCL9 ; determining
the level of expression of at least one gene selected from the
group consisting of Foxp3 , 112, Ilia, Illb, C3 , S100a8,
S100a9, Cxcl2, Cxcl3 , Ccl4, Ccl3 and Cdl4; determining the
level of expression of at least one gene selected from the
group consisting of the genes presented in Table 8 ;
determining the level of expression of at least one gene
selected from the group consisting of the genes presented in
Table 10; determining the level of expression of at least one
gene selected from the group consisting of FoxP3 , GPR83 , CD14,
TLR2, IFNG, CD40 and ILlB; determining the level of expression
of at least one gene selected from the group consisting of the
genes presented in Table 12; or determining gene set
enrichment analysis for genes associated with at least one
cell type selected from the group consisting of FoxP3+ CD4+ T
cells, CD4+ T cells CD8+ T cells, gamma delta T cells, natural
killer T cells, CD4+ CD8+ T cells, macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells, dendritic
cells, fibroblastic reticular cells, fibroblasts and
granulocytes ,
thereby characterizing the glatiramer acetate related drug substance
or drug product of step a ) .
The present invention also provides a process for characterizing a
glatiramer acetate related drug substance or drug product comprising
the steps of:
a ) obtaining a batch of the glatiramer acetate related drug
substance or drug product;
b ) immunizing a mammal with a predetermined amount of a
glatiramer acetate related drug substance or drug product;
c ) preparing a culture of cells from the mammal of step b ) at a
predetermined time after immunization;
d ) incubating cells from the culture of step c ) with a
predetermined amount of the glatiramer acetate related drug
substance or drug product of step a ) ; and
e ) determining the level of biological activity of the cells of
step c ) selected from the group consisting of, immune response
to antigen presenting cells, differentiation of effector
lymphocytes, suppression of T lymphocytes, activation of Foxp3
positive regulatory T cells, expansion of mononuclear
leukocytes, proliferation of T lymphocytes, expansion of
lymphocytes, differentiation of naive lymphocytes,
inflammatory response, adhesion of immune cells, cell
movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever,
thereby characterizing the glatiramer acetate related drug substance
or drug product of step a ) .
The present invention also provides a process for discriminating
between glatiramer acetate related drug substances or drug products
comprising the steps of:
i ) characterizing two or more glatiramer acetate related drug
substances or drug products according to a process of the
present invention to obtain characteristics of each of the
glatiramer acetate related drug substances or drug products;
and
ii) comparing the characteristics of the glatiramer acetate
related drug substances or drug products obtained in step i ) ,
thereby discriminating between the glatiramer acetate related drug
substances or drug products .
The present invention also provides a process for producing a drug
product comprising a glatiramer acetate related drug substance, the
improvement comprising the steps of:
i ) characterizing the glatiramer acetate related drug substance
according to a process of the present invention, wherein step
e ) comprises determining the level of expression of one or
more genes selected from the group consisting of Ecml, Presl,
Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590, Gprll4, Tmie,
Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c, Als2cl, 2810410P22Rik,
Arl5a, Gbp2, Rasgrpl, Ankrd37, Tpil, 4930583Hl4Rik, Ifit3,
LOC667370, Klhdcl, Cd247, Igfbp4, Oas2, Bclllb, Fscnl, Ctsg,
Mpo, Prtn3, Lyzs, E rl , Chi311, Anxa3 , Hp, Lyz2 , Lyz, Ferll3,
Sirpa, Cd63, Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp,
S100a8, S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi ,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3 ,
Gpr84, Acta2, Lcn2 , Hmoxl , Tpsabl, Ccl4, 112, Inhba, Cxcll,
Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2, Ilia, Ccl3 ,
6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt, LOC385615 and
Scml4; determining the level of expression of one or more
genes selected from the group consisting of the genes
presented in Table 8 ; determining the level of expression of
one or more genes selected from the group consisting of the
genes presented in Table 10; determining the level of
expression of one or more genes selected from the group
consisting of FoxP3 , GPR83, CD14, TLR2 , IFNG, CD40 and IL1B;
determining the level of expression of one or more genes
selected from the group consisting of the genes presented in
Table 12 ,· or determining gene set enrichment analysis for
genes associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells CD8+ T
cells, gamma delta T cells, natural killer T cells, CD4+ CD8+
T cells, macrophage cells, monocyte cells stromal cells,
multi-lineage progenitor cells, dendritic cells, fibroblastic
reticular cells, fibroblasts and granulocytes; and;
discarding the batch of the glatiramer acetate related drug
substance as unacceptable for inclusion in the drug product if
the level of expression of a gene selected from the group
consisting of Ecml, Presl, Pdlim4, Gpr83, Ifng, 1124,
LOC100046608, Gm590, Gprll4, Tmie, Rasgrpl, Myo6 , Pfkp, Uspl8,
Arl4c, Als2cl, 2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37,
Tpil, 4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igfbp4,
Oas2 Bclllb, 6720418B0lRik, 5830496LllRik, Cd8bl , Fcgrt,
LOC385615 and Scml4 is decreased relative to a reference
standard or if the level of expression of a gene selected from
the group consisting of Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl,
Chi311, Anxa3, Hp, Lyz2, Lyz , Ferll3, Sirpa, Cd63, Clec4n,
Clec4d, EG433016, Stfal, Chi313 Ngp, S100a8, S100a9, Clecsf9,
Saa3, 5033414K04Rik, Slc7all, Slpi, Cdl4, Fpr2 , Fcgr3 , F10,
Gpnmb, Tgfbi, Mmpl4, Slcllal, C3 , Gpr84, Acta2 , Lcn2 , Hmoxl ,
Tpsabl, Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl, Gprl09a,
Gp38, Illb, Cxcl2, Ilia, and Ccl3, is increased relative to a
reference standard; discarding the batch of the glatiramer
acetate related drug substance as unacceptable for inclusion
in the drug product if the level of expression of a gene
selected from the group consisting of the genes presented in
Table 8 is not substantially identical to the level of
expression of a reference standard; discarding the batch of
the glatiramer acetate related drug substance as unacceptable
for inclusion in the drug product if the level of expression
of a gene selected from the group consisting of the genes
presented in Table 10 is not substantially identical to the
level of expression of a reference standard; discarding the
batch of the glatiramer acetate related drug substance as
unacceptable for inclusion in the drug product if the level
of expression of a gene selected from the group consisting of
GPR83, IFNG and Foxp3 is decreased or if the level of
expression of a gene selected from the group consisting of
CD14, CD40, TLR2 and ILlB is increased; discarding the batch
of the glatiramer acetate related drug substance as
unacceptable for inclusion in the drug product if the level of
expression of a gene selected from the group consisting of the
genes identified in Table 12 as FoxP3+ T cell genes is
decreased or if the level of expression of a gene selected
from the group consisting of the genes identified in Table 12
as macrophage genes and the genes identified in Table 12 as
monocyte genes is increased; or discarding the batch of the
glatiramer acetate related drug substance as unacceptable for
inclusion in the drug product if gene set enrichment analysis
indicates downregulation or a lack of upregulation for genes
associated with at least one cell type selected from the group
consisting of FoxP3+ CD4+ T cells, CD4+ T cells CD8+ T cells,
gamma delta T cells, natural killer T cells and CD4+ CD8+ T
cells or if gene set enrichment analysis indicates
upregulation or a lack of downregulation for genes associated
with at least one cell type selected from the group consisting
of macrophage cells, monocyte cells stromal cells, multilineage
progenitor cells, dendritic cells, fibroblastic
reticular cells, fibroblasts and granulocytes.
The present invention also provides a process for producing a drug
product comprising a glatiramer acetate related drug substance, the
improvement comprising the steps of:
i ) characterizing the glatiramer acetate related drug substance
according to a process of the present invention, wherein step
e ) comprises determining the level of expression of at least
one gene selected from the group consisting of Foxp3 , 112,
Ilia, Illb, C3, S100a8, Sl00a9, Cxcl2 , Cxcl3 , Ccl4, Ccl3 and
Cdl4;
ii) discarding the batch of the glatiramer acetate related drug
substance as unacceptable for inclusion in the drug product if
the level of expression of FoxP3 is decreased relative to a
reference standard or if the level of expression of at least
one gene selected from the group consisting of 112, Ilia,
Illb, C3, Sl00a8, S100a9, Cxcl2, Cxcl3, Ccl4, Ccl3 and Cdl4 is
increased relative to a reference standard.
The present invention also provides a process for producing a drug
product comprising a glatiramer acetate related drug substance, the
improvement comprising the steps of:
i ) characterizing the glatiramer acetate related drug substance
according to a process of the present invention, wherein step
e ) comprises determining the level of biological activity of
the cells of step c ) selected from the group consisting of,
immune response to antigen presenting cells, differentiation
of effector lymphocytes, suppression of T lymphocytes,
activation of Foxp3 positive regulatory T cells, expansion of
mononuclear leukocytes, proliferation of T lymphocytes,
expansion of lymphocytes, differentiation of naive
lymphocytes, inflammatory response, adhesion of immune cells,
cell movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever;
ii) discarding the batch of the glatiramer acetate related drug
substance as unacceptable for inclusion in the drug product if
the level of biological activity selected from the group
consisting of immune response to antigen presenting cells,
differentiation of effector lymphocytes, suppression of T
lymphocytes and activation of Foxp3 positive regulatory T
cells is decreased relative to a reference standard or if the
level of biological activity selected from the group
consisting of expansion of mononuclear leukocytes,
proliferation of T lymphocytes, expansion of lymphocytes,
differentiation of naive lymphocytes, inflammatory response,
adhesion of immune cells, cell movement, migration of cells,
chemotaxis of cells, cell movement of phagocytes, chemotaxis
of monocytes, cell movement of monocytes and fever is
increased relative to a reference standard.
The present invention also provides a process for releasing a drug
product comprising a glatiramer acetate related drug substance, the
improvement comprising the steps of:
i ) characterizing the glatiramer acetate related drug product
according to a process of the present invention, wherein step
e ) comprises determining the level of expression of one or
more genes selected from the group consisting of Ecml, Presl,
Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590, Gprll4, Tmie,
Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c, Als2cl, 2810410P22Rik,
Arl5a, Gbp2, Rasgrpl, Ankrd37, Tpil, 4930583Hl4Rik, Ifit3,
LOC667370, Klhdcl, Cd247, Igfbp4, 0as2, Bclllb, Fscnl, Ctsg,
Mpo, Prtn3, Lyzs , Emrl, Chi311, Anxa3 , Hp, Lyz2 , Lyz, Ferll3 ,
Sirpa, Cd63, Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp,
S100a8, Sl00a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3 ,
Gpr84, Acta2, Lcn2, Hmoxl, Tpsabl, Ccl4, 112, Inhba, Cxcll,
Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2 , Ilia, Ccl3,
6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt, LOC385615 and
Scml4; determining the level of expression of one or more
genes selected from the group consisting of the genes
presented in Table 8 ; determining the level of expression of
one or more genes selected from the group consisting of the
genes presented in Table 10; determining the level of
expression of one or more genes selected from the group
consisting of FoxP3 , GPR83 , CD14, TLR2 , IFNG, CD40 and ILlB;
determining the level of expression of one or more genes
selected from the group consisting of the genes presented in
Table 12; or determining gene set enrichment analysis for
genes associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells CD8+ T
cells, gamma delta T cells, natural killer T cells, CD4+ CD8+
T cells, macrophage cells, monocyte cells stromal cells,
multi-lineage progenitor cells, dendritic cells, fibroblastic
reticular cells, fibroblasts and granulocytes; and;
ii) discarding the batch of the glatiramer acetate related drug
product as unacceptable for release if the level of expression
of a gene selected from the group consisting of Ecml, Presl,
Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590, Gprll4, Tmie,
Rasgrpl, Myo6 , Pfkp, Uspl8, Arl4c, Als2cl, 2810410P22Rik,
Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil, 4930583Hl4Rik, Ifit3,
LOC667370, Klhdcl, Cd247, Igfbp4, Oas2 Bclllb, 6720418B0lRik,
5830496LllRik, Cd8bl, Fcgrt, LOC385615 and Scml4 is decreased
relative to a reference standard or if the level of expression
of a gene selected from the group consisting of Fscnl, Ctsg,
Mpo, Prtn3, Lyzs, E rl , Chi311, Anxa3 , Hp, Lyz2, Lyz , Ferll3,
Sirpa, Cd63, Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp,
S100a8, S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3 ,
Gpr84, Acta2, Lcn2 , Hmoxl , Tpsabl, Ccl4, 112, Inhba, Cxcll,
Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2, Ilia, and Ccl3, is
increased relative to a reference standard; discarding the
batch of the glatiramer acetate related drug product as
unacceptable for release if the level of expression of a gene
selected from the group consisting of the genes presented in
Table 8 is not substantially identical to the level of
expression of a reference standard; discarding the batch of
the glatiramer acetate related drug product as unacceptable
for release if the level of expression of a gene selected from
the group consisting of the genes presented in Table 10 is not
substantially identical to the level of expression of a
reference standard; discarding the batch of the glatiramer
acetate related drug product as unacceptable for release if
the level of expression of a gene selected from the group
consisting of GPR83, IFNG and Foxp3 is decreased or if the
level of expression of a gene selected from the group
consisting of CD14, CD40, TLR2 and ILlB is increased;
discarding the batch of the glatiramer acetate related drug
product as unacceptable for release if the level of expression
of a gene selected from the group consisting of the genes
identified in Table 12 as FoxP3+ T cell genes is decreased or
if the level of expression of a gene selected from the group
consisting of the genes identified in Table 12 as macrophage
genes and the genes identified in Table 12 as monocyte genes
is increased; or discarding the batch of the glatiramer
acetate related drug product as unacceptable for release if
gene set enrichment analysis indicates downregulation or a
lack of upregulation for genes associated with at least one
cell type selected from the group consisting of FoxP3+ CD4+ T
cells, CD4+ T cells CD8+ T cells, gamma delta T cells, natural
killer T cells and CD4+ CD8+ T cells or if gene set enrichment
analysis indicates upregulation or a lack of downregulation
for genes associated with at least one cell type selected from
the group consisting of macrophage cells, monocyte cells
stromal cells, multi-lineage progenitor cells, dendritic
cells, fibroblastic reticular cells, fibroblasts and
granulocytes .
The present invention also provides a process for releasing a drug
product comprising a glatiramer acetate related drug substance, the
improvement comprising the steps of:
i ) characterizing the glatiramer acetate related drug product
according to a process of the present invention, wherein step
e ) comprises determining the level of expression of at least
one gene selected from the group consisting of Foxp3 , 112,
Ilia, Illb, C3, S100a8, S100a9, Cxcl2, Cxcl3, Ccl4, Ccl3 and
Cdl4;
ii) discarding the batch of the glatiramer acetate related drug
product as unacceptable for release if the level of expression
of FoxP3 is decreased relative to a reference standard or if
the level of expression of at least one gene selected from the
group consisting of 112, Ilia, Illb, C3 , S100a8, S100a9,
Cxcl2, Cxcl3, Ccl4, Ccl3 and Cdl4 is increased relative to a
reference standard.
The present invention also provides a process for releasing a drug
product comprising a glatiramer acetate related drug substance, the
improvement comprising the steps of:
i ) characterizing the glatiramer acetate related drug product
according to a process of the present invention, wherein step
e ) comprises determining the level of biological activity of
the cells of step c ) selected from the group consisting of,
immune response to antigen presenting cells, differentiation
of effector lymphocytes, suppression of T lymphocytes,
activation of Foxp3 positive regulatory T cells, expansion of
mononuclear leukocytes, proliferation of T lymphocytes,
expansion of lymphocytes, differentiation of naive
lymphocytes, inflammatory response, adhesion of immune cells,
cell movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever;
ii) discarding the batch of the glatiramer acetate related drug
product as unacceptable for release if the level of biological
activity selected from the group consisting of immune response
to antigen presenting cells, differentiation of effector
lymphocytes, suppression of T lymphocytes and activation of
Foxp3 positive regulatory T cells is decreased relative to a
reference standard or if the level of biological activity
selected from the group consisting of expansion of mononuclear
leukocytes, proliferation of T lymphocytes, expansion of
lymphocytes, differentiation of naive lymphocytes,
inflammatory response, adhesion of immune cells, cell
movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever is increased relative to a reference
standard.
The present invention also provides a method of identifying
suboptimal activity of a glatiramer acetate related drug substance
or drug product comprising the steps of:
a ) administering a glatiramer acetate related drug substance or
drug product to a rodent;
b ) determining the level of biological activity of the rodent
selected from the group consisting of, immune response to
antigen presenting cells, differentiation of effector
lymphocytes, suppression of T lymphocytes, activation of Foxp3
positive regulatory T cells, expansion of mononuclear
leukocytes, proliferation of T lymphocytes, expansion of
lymphocytes, differentiation of naive lymphocytes,
inflammatory response, adhesion of immune cells, cell
movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever; and
c ) identifying the glatiramer acetate related drug substance or
drug product as causing a suboptimal activity if the level of
biological activity selected from the group consisting of
immune response to antigen presenting cells, differentiation
of effector lymphocytes, suppression of T lymphocytes and
activation of Foxp3 positive regulatory T cells is decreased
relative to a reference standard or if the level of biological
activity selected from the group consisting of expansion of
mononuclear leukocytes, proliferation of T lymphocytes,
expansion of lymphocytes, differentiation of naive
lymphocytes, inflammatory response, adhesion of immune cells,
cell movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever is increased relative to a reference
standard,
thereby identifying suboptimal activity of the glatiramer acetate
related drug substance or drug product .
The present invention also provides a method of identifying
suboptimal activity of a glatiramer acetate related drug substance
or drug product comprising the steps of:
a ) administering a glatiramer acetate related drug substance or
drug product to a rodent
b ) determining the level of expression in the rodent of one or
more genes selected from the group consisting of Ecml, Presl,
Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590, Gprll4, Tmie,
Rasgrpl, Myo6 , Pfkp, Uspl8, Arl4c, Als2cl, 2810410P22Rik,
Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil, 4930583H14Rik, Ifit3,
LOC667370, Klhdcl, Cd247, Igfbp4, Oas2, Bclllb, Fscnl, Ctsg,
Mpo, Prtn3, Lyzs, Emrl, Chi311, Anxa3 , Hp, Lyz2, Lyz, Ferll3,
Sirpa, Cd63, Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp,
S100a8, Sl00a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3,
Gpr84, Acta2, Lcn2, Hmoxl, Tpsabl, Ccl4, 112, Inhba, Cxcll,
Serpinb2, Uppl , Gprl09a, Gp38, Illb, Cxcl2, Ilia, Ccl3,
6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt , LOC385615 and
Scml4; determining the level of expression of one or more
genes selected from the group consisting of the genes
presented in Table 8 ; determining the level of expression of
one or more genes selected from the group consisting of the
genes presented in Table 10; determining the level of
expression of one or more genes selected from the group
consisting of FoxP3 , GPR83 , CD14, TLR2 , IFNG, CD40 and ILlB;
determining the level of expression of one or more genes
selected from the group consisting of the genes presented in
Table 12; or determining gene set enrichment analysis for
genes associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells CD8+ T
cells, gamma delta T cells, natural killer T cells, CD4+ CD8 +
T cells, macrophage cells, monocyte cells stromal cells,
multi-lineage progenitor cells, dendritic cells, fibroblastic
reticular cells, fibroblasts and granulocytes; and
identifying the glatiramer acetate related drug substance or
drug product as causing a suboptimal activity if the level of
expression of a gene selected from the group consisting of
Ecml, Presl, Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590,
Gprll4, Tmie, Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c, Als2cl,
2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil,
4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igfbp4, 0as2
Bclllb, 6720418B0lRik, 5830496LllRik, Cd8bl , Fcgrt, LOC385615
and Scml4 is decreased relative to a reference standard or if
the level of expression of a gene selected from the group
consisting of Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl, Chi311,
Anxa3, Hp, Lyz2, Lyz , Ferll3, Sirpa, Cd63, Clec4n, Clec4d,
EG433016, Stfal, Chi313 Ngp, S100a8, Sl00a9, Clecsf9, Saa3 ,
5033414K04Rik, Slc7all, Slpi, Cdl4, Fpr2, Fcgr3, F10, Gpnmb,
Tgfbi, Mmpl4, Slcllal, C3 , Gpr84, Acta2, Lcn2, Hmoxl, Tpsabl,
Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb,
Cxcl2, Ilia, and Ccl3, is increased relative to a reference
standard; identifying the glatiramer acetate related drug
substance or drug product as causing a suboptimal activity if
the level of expression of a gene selected from the group
consisting of the genes presented in Table 8 is not
subs an ia y identical to the level of expression of a
reference standard; identifying the glatiramer acetate related
drug substance or drug product as causing a suboptimal
activity if the level of expression of a gene selected from
the group consisting of the genes presented in Table 10 is not
substantially identical to the level of expression of a
reference standard; identifying the glatiramer acetate related
drug substance or drug product as causing a suboptimal
activity if the level of expression of a gene selected from
the group consisting of GPR83 , IFNG and Foxp3 is decreased or
if the level of expression of a gene selected from the group
consisting of CD14, CD40, TLR2 and ILlB is increased;
identifying the glatiramer acetate related drug substance or
drug product as causing a suboptimal activity if the level of
expression of a gene selected from the group consisting of the
genes identified in Table 12 as FoxP3+ t cell genes is
decreased or if the level of expression of a gene selected
from the group consisting of the genes identified in Table 12
as macrophage genes and the genes identified in Table 12 as
monocyte genes is increased; or identifying the glatiramer
acetate related drug substance or drug product as causing a
suboptimal activity if gene set enrichment analysis indicates
downregulation or a lack of upregulation for genes associated
with at least one cell type selected from the group consisting
of FoxP3+ CD4+ T cells, CD4+ T cells CD8+ T cells, gamma delta
T cells, natural killer T cells and CD4+ CD8+ T cells or if
gene set enrichment analysis indicates upregulation or a lack
of downregulation for genes associated with at least one cell
type selected from the group consisting of macrophage cells,
monocyte cells stromal cells, multi-lineage progenitor cells,
dendritic cells, fibroblastic reticular cells, fibroblasts and
granulocytes ,
thereby identifying suboptimal activity of the glatiramer acetate
related drug substance or drug product .
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 : PCA of quantile-normalized, batch-corrected signals,
colored by activation groups .
Figure 2 : Gene-wise hierarchical clustering of 1474 genes with FDRadjusted
p value < 0.05 and fold change ³ 1.3 between GA and Mediumreactivated
samples . The genes and gene symbols are listed in
columns, and the samples, ordered by sample type, are listed in
rows .
Figure 3 : Gene-wise hierarchical clustering of 98 genes with FDRadjusted
p value < 0.05 and fold change >1.3 between GA-RS and 8 GANatco
samples. The genes and gene symbols are listed in rows, and
the samples, ordered by sample type, are listed in columns. The
expression levels of Medium and GA-DP samples for these 98 genes are
presented as well.
Figure 4 : The biological impact of GA is significantly more
consistent than that of other glatiramoids . Among probes with
variability induced by activation, more than 4-fold higher probes
had significant variation by F-test in other glatiramoid-activated
samples when compared to GA activated samples (A) . Defining
tolerance as the percentage of samples with expression levels
falling within the range between the maximum and minimum expression
levels induced by reference standard for that probe, for any given
tolerance threshold the number of probes failing to meet this this
threshold is displayed for both the other glatiramoid and GA (B) ,
showing that in almost all cases more probes fail to meet tolerance
following induction by the other glatiramoid. For each individual
other glatiramoid batch, the percentage of probes with significant
differences in variability when compared to either GA or GA
reference standard are plotted in C , along with the percentage of
probes with differences in variability between GA and reference
standard (dashed green line) for comparison. In each case the other
glatiramoid batches have greater differences in variability.
Figure 5 : Plot of the coefficient of variation (CV) as a function of
intensity for each of the probes when activated by other glatiramoid
(black) and GA (red) , showing the smaller range of CVs in GA and the
wider range in other glatiramoids at any given intensity.
Figure 6 : GA induces Tregs more effectively than other glatiramoids .
(A) GA induces significantly higher expression of FoxP3 than other
glatiramoid. FoxP3 is a key marker of Tregs, and (B) another key
Treg marker Gpr83 shows a similar pattern of expression. (C) Both
FoxP3 and Gpr83 are low in the same samples as indicated by scatter
plot, further strengthening the case that the other glatiramoid
fails to induce a strong Treg response in some patients. (D) As
further evidence of the difference in FoxP3 induction, GSEA analysis
found a significantly stronger upregulation of FoxP3 target genes in
GA-activated samples than in other glatiramoid-activated samples.
(E) GSEA analysis also found a significant enrichment of Tregspecific
genes among the genes with higher expression in GA than in
other glatiramoid. NS = not significant .
Figure 7 : The GSEA enrichment plots for the FoxP3 and Treg GSEA
analyses reported in Figure 6D-E.
Figure 8 : Cell-type specific differences in the biological impact of
GA and other glatiramoids. The heat map depicts relative expression
of specific genes in GA-activated samples and other glatiramoidactivated
samples . Each of the rows within the Treg section
represents a gene with a high cell-type specificity scores for
Tregs, while each of the rows in the macrophages and monocyte
sections represents genes with high cell-type specificity scores for
each of those cell types. The associated gene lists appear as
supplementary information. Overall, GA induces higher expression of
Treg-associated genes than other glatiramoid, while other
glatiramoid induces higher expression of macrophage and monocyteassociated
genes than GA.
Figure 9 : Box plots of CD14 and TLR2 , depicting the lower expression
levels in GA and Reference compared to other glatiramoid. This is an
additional way of visualizing the differences depicted by kernel
density plots in Figure 10A.
Figure 10: Other glatiramoid' s impact on monocytes may differ from
GA's impact. (A) Other glatiramoid induces significantly higher
expression of CD14 and TLR2, as determined by a Wilcoxon rank sum
test and depicted as kernel density plots, which can be likened to a
smoothed histogram. (B) CD14 and TLR2 expression are both unusually
high in the same (mostly other glatiramoid) samples. (C) FoxP3
expression is unusually low in the sample samples in which CD14
expression is unusually high, suggesting that the other
glatiramoid' s different impact on monocytes may be related to its
different impact on Tregs and consistent with literature suggesting
that monocytes play a role in GA-induced FoxP3 expression. (D) FoxP3
expression is unusually low in the sample samples in which ILlB
expression is unusually high, suggesting that the other
glatiramoid' s different impact on monocytes may be related to the
differences between LPS-activated monocytes and T-cell contact
activated monocytes, which have been described in the literature as
having opposite impacts on ILlB production. (E) GSEA analysis found
a significant enrichment of monocyte and macrophage -specific genes
among the genes with higher expression in other glatiramoid than GA.
NS = not significant .
Figure 11: Scatter plots showing that the same other glatiramoid
samples with unusually low FoxP3 expression also had unusually low
IFNG expression, by two different probes of IFNG. Scatter plots
illustrating that for two different probes of IFNG, GA and Reference
standard upregulated IFNG to a greater extent than other glatiramoid
did.
Figure 12: Kernel density plot of CD40, illustrating the fact that
this gene had higher expression in other glatiramoid-activated
samples than in GA activated samples, consistent with the
determination by the Wilcoxon rank-sum test and consistent with
literature .
Figure 13 : Scatterplot illustrating the high degree of correlation
between CD14 and ILlB, lending support to the hypothesis that the
ILlB is expressed primarily by monocytes.
Figure 14: GSEA analysis showing that genes with higher expression
in other glatiramoid than medium are enriched in genes specific to
CDl6dim monocytes, while genes with higher expression in GA than
medium are enriched in genes specific to CD16+ monocytes.
Figure 15: Flow chart of process for comparing an innovative
medicine to a other glatiramoid, and model of key differences
between GA and other glatiramoid (A) Oveview of the methods for
analyzing gene expression data to compare the immunological impact
of GA to that of other glatiramoid. After processing, direct
differences are identified by multiple parametric methods, nonparametric
methods, as well as ANOVA-based pattern analysis, and
variability analysis. The genes identified by these methods are
analyzed using a variety of enrichment -based methods, which result
in hypotheses that are then verified through additional methods. (B)
The key hypotheses emerging from our studies involve the greater
heterogeneity in the other glatiramoid' s biological impact compared
to GA's, and the fact that GA appears to more effectively upregulate
FoxP3 expression and promote tolerance-inducing Tregs, while other
glatiramoid appears to upregulated myeloid lineage cells such as
monocytes and macrophages which may impair tolerance. Given these
findings, it is reasonable to hypothesize that GA may suppress
harmful cytotoxic cells more effectively than other glatiramoid, and
this hypothesis warrants further investigation.
Figure 16: Illustration of the tolerance method for comparing
variability. The expression of genes following activation by GA and
other glatiramoid are assessed to determine the percentage of
samples following within a tolerance defined by the maximum and
minimum expression levels induced by the reference standard (top and
bottom of the red box for Gpr83 , left and right sides of the red box
for FoxP3 ).
DETAILED DESCRIPTION OF THE INVENTION
Embodiments of the Inven on
The present invention provides a process for characterizing a
glatiramer acetate related drug substance or drug product comprising
the steps of :
a ) obtaining a batch of the glatiramer acetate related drug
substance or drug product;
b ) immunizing a mammal with a predetermined amount of a
glatiramer acetate related drug substance or drug product;
c ) preparing a culture of cells from the mammal of step b ) at a
predetermined time after immunization;
d ) incubating cells from the culture of step c ) with a
predetermined amount of the glatiramer acetate drug related
substance or drug product of step a ) ; and
e ) determining the level of expression of at least one gene
selected from the group consisting of genes regulated by
glatiramer acetate reference standard or glatiramer acetate
drug substance or drug product in Gene Expression Omnibus
accession number GSE40566; determining the level of expression
of at least one gene selected from the group consisting of
Ecml, Presl, Pdlim4, Gpr83, Ifng, 1124, LOC100046608 , Gm590,
Gprll4, Tmie, Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c, Als2cl,
2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil,
4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igfbp4, 0as2,
Bclllb, Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl, Chi311, Anxa3 ,
Hp, Lyz2, Lyz, Ferll3 , Sirpa, Cd63, Clec4n, Clec4d, EG433016,
Stfal, Chi313 Ngp, S100a8, S100a9, Clecsf9, Saa3 ,
5033414K04Rik, Slc7all, Slpi, Cdl4, Fpr2 , Fcgr3 , F10, Gpnmb,
Tgfbi, Mmpl4, Slcllal, C3 , Gpr84, Acta2 , Lcn2, Hmoxl , Tpsabl,
Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb,
Cxcl2, Ilia, Ccl3, 6720418B0lRik, 5830496LllRik, Cd8bl , Fcgrt ,
LOC385615 and Scml4; determining the level of expression of at
least one gene selected from the group consisting of CD40,
CD86, G 3 , HLA-DMA, HLA-DMB, ICOS , IFNG, IFNGR2 , IL2 , IL13 ,
IL4, IL18, IL12RB1, IL17A, IL17F, IL18R1, IL2RA, IL2RG, IL4R,
IL6R, TBX21, TGFBR2 , TNF, FOXP3 , IL10RB, KLRDl , CD69, LTB ,
CD83, PRFl, CAMK2D, LTA, FSCN1 , TLR7 , CSF2 , CCR7 , FASLG, ILIA,
CCL5, CD8B, CXCL10, TLR2 , CCL4 , TLR7 , IGHAl , IL24, SOCSl,
OAS1, JAK1, PTPN2, IFITMl , IFI35, S A 2 , BCL2 , MVD, FDPS,
SQLE, NSDHL, DHCR24, Acat2/Acat3, MSMOl , LSS, CYP51A1, NFKBIE,
PIK3R1, PPP3CC, CD3D, IL2RB, PTEN, CD3G, ICOS, CAMK2D, NFAT5 ,
LAT, ITK, H2-M2, FASLG, LIF, IGHAl, PRKACB , SGKl , MAPK11,
TSC22D3 , JU , FKBP5 , ADRB2 , MAP3K1 , MAPK12, POU2F1 , SMARCA2 ,
CDKNlA, TGFB3, HSP90AA1, DHCR24, CCR5 , and CXCL9 ; determining
the level of expression of at least one gene selected from the
group consisting of Foxp3 , 112, Ilia, Illb, C3 , S100a8,
S100a9, Cxcl2, Cxcl3, Ccl4, Ccl3 and Cdl4; determining the
level of expression of at least one gene selected from the
group consisting of the genes presented in Table 8 ;
determining the level of expression of at least one gene
selected from the group consisting of the genes presented in
Table 10; determining the level of expression of at least one
gene selected from the group consisting of FoxP3 , GPR83, CD14,
TLR2, IFNG, CD40 and ILlB; determining the level of expression
of at least one gene selected from the group consisting of the
genes presented in Table 12; or determining gene set
enrichment analysis for genes associated with at least one
cell type selected from the group consisting of FoxP3+ CD4+ T
cells, CD4+ T cells CD8+ T cells, gamma delta T cells, natural
killer T cells, CD4+ CD8+ T cells, macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells, dendritic
cells, fibroblastic reticular cells, fibroblasts and
granulocytes ,
thereby characterizing the glatiramer acetate related drug substance
or drug product of step a ) .
The present invention also provides a process for characterizing a
glatiramer acetate related drug substance or drug product comprising
the steps of:
a ) obtaining a batch of the glatiramer acetate related drug
substance or drug product;
b ) immunizing a mammal with a predetermined amount of a
glatiramer acetate related drug substance or drug product;
c ) preparing a culture of cells from the mammal of step b ) at a
predetermined time after immunization;
d ) incubating cells from the culture of step c ) with a
predetermined amount of the glatiramer acetate related drug
substance or drug product of step a ) ; and
e ) determining the level of biological activity of the cells of
step c ) selected from the group consisting of, immune response
to antigen presenting cells, differentiation of effector
lymphocytes, suppression of T lymphocytes, activation of Foxp3
positive regulatory T cells, expansion of mononuclear
leukocytes, proliferation of T lymphocytes, expansion of
lymphocytes, differentiation of naive lymphocytes,
inflammatory response, adhesion of immune cells, cell
movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell movement
of monocytes and fever,

claimed:
1 . A process for characterizing a glatiramer acetate related drug
substance or drug product comprising the steps of:
a ) obtaining a batch of the glatiramer acetate related drug
substance or drug product;
b ) immunizing a mammal with a predetermined amount of a
glatiramer acetate related drug substance or drug
product;
c ) preparing a culture of cells from the mammal of step b )
at a predetermined time after immunization;
d ) incubating cells from the culture of step c ) with a
predetermined amount of the glatiramer acetate drug
related substance or drug product of step a ) ; and
e ) determining the level of expression of at least one gene
selected from the group consisting of genes regulated by
glatiramer acetate drug substance or drug product in Gene
Expression Omnibus accession number GSE40566; determining
the level of expression of at least one gene selected
from the group consisting of Ecml, Presl, Pdlim4, Gpr83,
Ifng, 1124, LOC100046608 , Gm590, Gprll4, Tmie, Rasgrpl,
Myo6, Pfkp, Uspl8, Arl4c, Als2cl, 2810410P22Rik, Arl5a,
Gbp2, Rasgrpl, Ankrd37, Tpil, 4930583Hl4Rik, Ifit3,
LOC667370, Klhdcl, Cd247, Igfbp4, 0as2, Bclllb, Fscnl,
Ctsg, Mpo, Prtn3 , Lyzs, Emrl, Chi311, Anxa3 , Hp, Lyz2,
Lyz, Ferll3, Sirpa, Cd63, Clec4n, Clec4d, EG433016,
Stfal, Chi313 Ngp, S100a8, S100a9, Clecsf9, Saa3,
5033414K04Rik, Slc7all, Slpi, Cdl4, Fpr2 , Fcgr3, F10,
Gpnmb, Tgfbi, Mmpl4, Slcllal, C3, Gpr84, Acta2, Lcn2,
Hmoxl, Tpsabl, Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl,
Gprl09a, Gp38, Illb, Cxcl2, Ilia, Ccl3, 6720418B0lRik,
5830496LllRik, Cd8bl , Fcgrt, LOC385615 and Scml4;
determining the level of expression of at least one gene
selected from the group consisting of CD40, CD86, GA A3 ,
HLA-DMA, HLA-DMB, ICOS, IFNG, IFNGR2 , IL2, IL13, IL4 ,
IL18, IL12RB1, IL17A, IL17F, IL18R1, IL2RA, IL2RG, IL4R,
IL6R, TBX21, TGFBR2 , TNF , FOXP3 , ILlORB, KLRDl, CD69,
LTB, CD83, PRF1, CAMK2D, LTA, FSCNl , TLR7 , CSF2 , CCR7 ,
FASLG, ILIA, CCL5 , CD8B, CXCL10, TLR2 , CCL , TLR7 , IGHAl ,
IL24, S0CS1, OAS1, JAKl , PTPN2 , IFITMl, IFI35, STAT2 ,
BCL2, MVD, FDPS, SQLE, NSDHL, DHCR24, Acat2/Acat3, MSMOl ,
LSS, CYP51A1, NFKBIE, PIK3R1, PPP3CC , CD3D, IL2RB, PTEN,
CD3G, ICOS, CAM 2D , NFA 5 , LAT, I K , H2-M2, FASLG, LIF,
IGHAl, PRKACB , SGKl , MAPKll, TSC22D3, U , FKBP5 , ADRB2 ,
MAP3K1, MAPK12, POU2F1, SMARCA2 , CDKNlA, TGFB3 , HSP90AA1,
DHCR24, CCR5, and CXCL9 ; determining the level of
expression of at least one gene selected from the group
consisting of Foxp3 , 112, Ilia, Illb, C3 , S100a8, S100a9,
Cxcl2, Cxcl3, Ccl4, Ccl3 and Cdl4; determining the level
of expression of at least one gene selected from the
group consisting of the genes presented in Table 8 ;
determining the level of expression of at least one gene
selected from the group consisting of the genes presented
in Table 10; determining the level of expression of at
least one gene selected from the group consisting of
FoxP3, GPR83, CD14 , TLR2 , IFNG, CD40 and ILlB;
determining the level of expression of at least one gene
selected from the group consisting of the genes presented
in Table 12; or determining gene set enrichment analysis
for genes associated with at least one cell type selected
from the group consisting of FoxP3+ CD4+ T cells, CD4+ T
cells CD8+ T cells, gamma delta T cells, natural killer T
cells, CD4+ CD8+ T cells, macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells,
dendritic cells, fibroblastic reticular cells,
fibroblasts and granulocytes,
thereby characterizing the glatiramer acetate related drug
substance or drug product of step a ) .
2 . A process for characterizing a glatiramer acetate related drug
substance or drug product comprising the steps of:
a ) obtaining a batch of the glatiramer acetate related drug
substance or drug product;
b ) immunizing a mammal with a predetermined amount of a
glatiramer acetate related drug substance or drug
product;
c ) preparing a culture of cells from the mammal of step b )
at a predetermined time after immunization;
d ) incubating cells from the culture of step c ) with a
predetermined amount of the glatiramer acetate related
drug substance or drug product of step a ) ; and
e ) determining the level of biological activity of the cells
of step c ) selected from the group consisting of, immune
response to antigen presenting cells, differentiation of
effector lymphocytes, suppression of T lymphocytes,
activation of Foxp3 positive regulatory T cells,
expansion of mononuclear leukocytes, proliferation of T
lymphocytes, expansion of lymphocytes, differentiation of
naive lymphocytes, inflammatory response, adhesion of
immune cells, cell movement, migration of cells,
chemotaxis of cells, cell movement of phagocytes,
chemotaxis of monocytes, cell movement of monocytes and
fever,
thereby characterizing the glatiramer acetate related drug
substance or drug product of step a ) .
3 . A process for discriminating between glatiramer acetate
related drug substances or drug products comprising the steps
of:
i ) characterizing two or more glatiramer acetate related
drug substances or drug products according to the process
of claim 1 or claim 2 to obtain characteristics of each
of the glatiramer acetate related drug substances or drug
products; and
ii) comparing the characteristics of the glatiramer acetate
related drug substances or drug products obtained in step
i ),
thereby discriminating between the glatiramer acetate related
drug substances or drug products .
4 . The process of any one of claims 1-3, wherein the mammal is a
rodent .
5 . The process of any one of claims 1-4, wherein the culture of
step c ) is a primary culture.
6 . The process of any one of claims 1-5, wherein the glatiramer
acetate related drug substance or drug product of step a ) is
glatiramer acetate drug substance or drug product .
7 . The process of any one of claims 1-5, wherein the glatiramer
acetate related drug substance or drug product of step a ) is a
glatiramer acetate related drug substance or drug product
other than glatiramer acetate drug substance or drug product .
8 . The process of any one of claims 1-7, wherein the glatiramer
acetate related drug substance or drug product of step b ) is
glatiramer acetate drug substance or drug product .
9 . The process of any one of claims 1-7, wherein the glatiramer
acetate related drug substance or drug product of step b ) is a
glatiramer acetate related drug substance or drug product
other than glatiramer acetate drug substance or drug product .
10. The process of any one of claims 1-7, wherein the glatiramer
acetate related drug substance or drug product of step b ) is
the same glatiramer acetate related drug substance or drug
product of step a ) .
11. The process of any one of claims 1-7, wherein the glatiramer
acetate related drug substance or drug product of step b ) is a
different glatiramer acetate related drug substance or drug
product than the glatiramer acetate related drug substance or
drug product of step a ) .
12. In a process for producing a drug product comprising a
glatiramer acetate related drug substance, the improvement
comprising the steps of :
i ) characterizing the glatiramer acetate related drug
substance according to the process of claim 1 , wherein
step e ) comprises determining the level of expression of
one or more genes selected from the group consisting of
Ecml, Presl, Pdlim4, Gpr83, Ifng, 1124, LOC100046608 ,
Gm590, Gprll4, ie , Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c,
Als2cl, 2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37,
Tpil, 4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247,
Igfbp4, Oas2, Bclllb, Fscnl, Ctsg, Mpo, Prtn3 , Lyzs,
Emrl, Chi311, Anxa3 , Hp, Lyz2, Lyz, Ferll3, Sirpa, Cd63,
Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp, S100a8,
S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3, F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3,
Gpr84, Acta2, Lcn2 , Hmoxl , Tpsabl, Ccl4, 112, Inhba,
Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2, Ilia,
Ccl3, 6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt,
LOC385615 and Scml4; determining the level of expression
of one or more genes selected from the group consisting
of the genes presented in Table 8 ; determining the level
of expression of one or more genes selected from the
group consisting of the genes presented in Table 10;
determining the level of expression of one or more genes
selected from the group consisting of FoxP3 , GPR83, CD14,
TLR2, IFNG, CD40 and ILlB; determining the level of
expression of one or more genes selected from the group
consisting of the genes presented in Table 12; or
determining gene set enrichment analysis for genes
associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells
CD8+ T cells, gamma delta T cells, natural killer T
cells, CD4+ CD8+ T cells, macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells,
dendritic cells, fibroblastic reticular cells,
fibroblasts and granulocytes; and;
discarding the batch of the glatiramer acetate related
drug substance as unacceptable for inclusion in the drug
product if the level of expression of a gene selected
from the group consisting of Ecml, Presl, Pdlim4, Gpr83,
Ifng, 1124, LOC100046608 , Gm590, Gprll4, Tmie, Rasgrpl,
Myo6, Pfkp, Uspl8, Arl4c, Als2cl, 2810410P22Rik, Arl5a,
Gbp2, Rasgrpl, Ankrd37, Tpil, 4930583Hl4Rik, Ifit3,
LOC667370, Klhdcl, Cd247, Igfbp4, Oas2 Bclllb,
6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt, LOC385615 and
Scml4 is decreased relative to a reference standard or if
the level of expression of a gene selected from the group
consisting of Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl,
Chi311, Anxa3, Hp, Lyz2, Lyz , Ferll3, Sirpa, Cd63,
Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp, S100a8,
S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3 ,
Gpr84, Acta2, Lcn2 , Hmoxl, Tpsabl, Ccl4, 112, Inhba,
Cxcll, Serpinb2, Up l , Gprl09a, Gp38, Illb, Cxcl2 , Ilia,
and Ccl3 , is increased relative to a reference standard;
discarding the batch of the glatiramer acetate related
drug substance as unacceptable for inclusion in the drug
product if the level of expression of a gene selected
from the group consisting of the genes presented in Table
8 is not substantially identical to the level of
expression of a reference standard; discarding the batch
of the glatiramer acetate related drug substance as
unacceptable for inclusion in the drug product if the
level of expression of a gene selected from the group
consisting of the genes presented in Table 10 is not
substantially identical to the level of expression of a
reference standard; discarding the batch of the
glatiramer acetate related drug substance as unacceptable
for inclusion in the drug product if the level of
expression of a gene selected from the group consisting
of GPR83, IFNG and Foxp3 is decreased or if the level of
expression of a gene selected from the group consisting
of CD14, CD40, TLR2 and ILlB is increased; discarding the
batch of the glatiramer acetate related drug substance as
unacceptable for inclusion in the drug product if the
level of expression of a gene selected from the group
consisting of the genes identified in Table 12 as FoxP3+
T cell genes is decreased or if the level of expression
of a gene selected from the group consisting of the genes
identified in Table 12 as macrophage genes and the genes
identified in Table 12 as monocyte genes is increased; or
discarding the batch of the glatiramer acetate related
drug substance as unacceptable for inclusion in the drug
product if gene set enrichment analysis indicates
downregulation or a lack of upregulation for genes
associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells
CD8+ T cells, gamma delta T cells, natural killer T cells
and CD4+ CD8+ T cells or if gene set enrichment analysis
indicates upregulation or a lack of downregulation for
genes associated with at least one cell type selected
from the group consisting of macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells,
dendritic cells, fibroblastic reticular cells,
fibroblasts and granulocytes .
In a process for producing a drug product comprising a
glatiramer acetate related drug substance, the improvement
comprising the steps of:
i ) characterizing the glatiramer acetate related drug
substance according to the process of claim 1 , wherein
step e ) comprises determining the level of expression of
at least one gene selected from the group consisting of
Foxp3, 112, Ilia, Illb, C3 , S100a8, S100a9, Cxcl2, Cxcl3,
Ccl4, Ccl3 and Cdl4;
ii) discarding the batch of the glatiramer acetate related
drug substance as unacceptable for inclusion in the drug
product if the level of expression of FoxP3 is decreased
relative to a reference standard or if the level of
expression of at least one gene selected from the group
consisting of 112, Ilia, Illb, C3 , Sl00a8, S100a9, Cxcl2,
Cxcl3, Ccl4, Ccl3 and Cdl4 is increased relative to a
reference standard.
In a process for producing a drug product comprising a
glatiramer acetate related drug substance, the improvement
comprising the steps of:
i ) characterizing the glatiramer acetate related drug
substance according to the process of claim 2 , wherein
step e ) comprises determining the level of biological
activity of the cells of step c ) selected from the group
consisting of, immune response to antigen presenting
cells, differentiation of effector lymphocytes,
suppression of T lymphocytes, activation of Foxp3
positive regulatory T cells, expansion of mononuclear
leukocytes, proliferation of T lymphocytes, expansion of
lymphocytes, differentiation of naive lymphocytes,
inflammatory response, adhesion of immune cells, cell
movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell
movement of monocytes and fever;
ii) discarding the batch of the glatiramer acetate related
drug substance as unacceptable for inclusion in the drug
product if the level of biological activity selected from
the group consisting of immune response to antigen
presenting cells, differentiation of effector
lymphocytes, suppression of T lymphocytes and activation
of Foxp3 positive regulatory T cells is decreased
relative to a reference standard or if the level of
biological activity selected from the group consisting of
expansion of mononuclear leukocytes, proliferation of T
lymphocytes, expansion of lymphocytes, differentiation of
naive lymphocytes, inflammatory response, adhesion of
immune cells, cell movement, migration of cells,
chemotaxis of cells, cell movement of phagocytes,
chemotaxis of monocytes, cell movement of monocytes and
fever is increased relative to a reference standard.
In a process for releasing a drug product comprising a
glatiramer acetate related drug substance, the improvement
comprising the steps of:
i ) characterizing the glatiramer acetate related drug
product according to the process of claim 1 , wherein step
e ) comprises determining the level of expression of one
or more genes selected from the group consisting of Ecml,
Presl, Pdlim4 , Gpr83 , Ifng, 1124, LOC100046608 , Gm590,
Gprll4, Tmie, Rasgrpl, Myo6 , Pfkp, Uspl8, Arl4c, Als2cl,
2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil,
4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igfbp4,
Oas2, Bclllb, Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl,
Chi311, Anxa3, Hp, Lyz2, Lyz , Ferll3 , Sirpa, Cd63 ,
Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp, S100a8,
S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgf i , Mmpl4, Slcllal, C3 ,
Gpr84, Acta2, Lcn2, Hmoxl, Tpsabl, Ccl4, 112, Inhba,
Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2, Ilia,
Ccl3, 6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt,
LOC385615 and Scml4; determining the level of expression
of one or more genes selected from the group consisting
o f the genes presented in Table 8 ; determining the level
o f expression o f one or more genes selected from the
group consisting o f the genes presented in Table 10;
determining the level of expression of one or more genes
selected from the group consisting of FoxP3 , GPR83 , CD14,
TLR2, IFNG, CD40 and ILlB; determining the level of
expression of one or more genes selected from the group
consisting o f the genes presented in Table 12; or
determining gene set enrichment analysis for genes
associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells
CD8+ T cells, gamma delta T cells, natural killer T
cells, CD4+ CD8+ T cells, macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells,
dendritic cells, fibroblastic reticular cells,
fibroblasts and granulocytes; and;
discarding the batch o f the glatiramer acetate related
drug product as unacceptable for release if the level of
expression of a gene selected from the group consisting
o f Ecml, Presl, Pdlim4, Gpr83 , Ifng, 1124, LOC100046608 ,
Gm590, Gprll4, Tmie, Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c,
Als2cl, 2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37,
Tpil, 4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247,
Igfbp4, 0as2 Bclllb, 6720418B0lRik, 5830496LllRik, Cd8bl ,
Fcgrt, LOC385615 and Scml4 is decreased relative to a
reference standard or if the level of expression of a
gene selected from the group consisting of Fscnl, Ctsg,
Mpo, Prtn3 , Lyzs , Emrl , Chi311 , Anxa3 , Hp, Lyz2 , Lyz ,
Ferll3, Sirpa, Cd63, Clec4n, Clec4d, EG433016, Stfal,
Chi313 Ngp, Sl00a8, Sl00a9, Clecsf9, Saa3, 5033414K04Rik,
Slc7all, Slpi, Cdl4, Fpr2 , Fcgr3 , FlO, Gpnmb, Tgfbi,
Mmpl4, Slcllal, C3, Gpr84, Acta2, Lcn2, Hmoxl, Tpsabl,
Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl, Gprl09a, Gp38,
Illb, Cxcl2, Ilia, and Ccl3, is increased relative to a
reference standard; discarding the batch of the
glatiramer acetate related drug product as unacceptable
for release if the level of expression of a gene selected
from the group consisting of the genes presented in Table
8 is not substantially identical to the level of
expression of a reference standard; discarding the batch
of the glatiramer acetate related drug product as
unacceptable for release if the level of expression of a
gene selected from the group consisting of the genes
presented in Table 10 is not substantially identical to
the level of expression of a reference standard;
discarding the batch of the glatiramer acetate related
drug product as unacceptable for release if the level of
expression of a gene selected from the group consisting
of GPR83, IFNG and Foxp3 is decreased or if the level of
expression of a gene selected from the group consisting
of CD14, CD40, TLR2 and IL1B is increased; discarding the
batch of the glatiramer acetate related drug product as
unacceptable for release if the level of expression of a
gene selected from the group consisting of the genes
identified in Table 12 as FoxP3+ T cell genes is
decreased or if the level of expression of a gene
selected from the group consisting of the genes
identified in Table 12 as macrophage genes and the genes
identified in Table 12 as monocyte genes is increased; or
discarding the batch of the glatiramer acetate related
drug product as unacceptable for release if gene set
enrichment analysis indicates downregulation or a lack of
upregulation for genes associated with at least one cell
type selected from the group consisting of FoxP3+ CD4+ T
cells, CD4+ T cells CD8+ T cells, gamma delta T cells,
natural killer T cells and CD4+ CD8+ T cells or if gene
set enrichment analysis indicates upregulation or a lack
of downregulation for genes associated with at least one
cell type selected from the group consisting of
macrophage cells, monocyte cells stromal cells, multilineage
progenitor cells, dendritic cells, fibroblastic
reticular cells, fibroblasts and granulocytes.
In a process for releasing a drug product comprising a
glatiramer acetate related drug substance, the improvement
comprising the steps of:
i ) characterizing the glatiramer acetate related drug
product according to the process of claim 1 , wherein step
e ) comprises determining the level of expression of at
least one gene selected from the group consisting of
Foxp3, 112, Ilia, Illb, C3 , S100a8, Sl00a9, Cxcl2, Cxcl3,
Ccl4, Ccl3 and Cdl4;
ii) discarding the batch of the glatiramer acetate related
drug product as unacceptable for release if the level of
expression of FoxP3 is decreased relative to a reference
standard or if the level of expression of at least one
gene selected from the group consisting of 112, Ilia,
Illb, C3, Sl00a8, Sl00a9, Cxcl2 , Cxcl3, Ccl4, Ccl3 and
Cdl4 is increased relative to a reference standard.
In a process for releasing a drug product comprising a
glatiramer acetate related drug substance, the improvement
comprising the steps of:
i ) characterizing the glatiramer acetate related drug
product according to the process of claim 2 , wherein step
e ) comprises determining the level of biological activity
of the cells of step c ) selected from the group
consisting of, immune response to antigen presenting
cells, differentiation of effector lymphocytes,
suppression of T lymphocytes, activation of Foxp3
positive regulatory T cells, expansion of mononuclear
leukocytes, proliferation of T lymphocytes, expansion of
lymphocytes, differentiation of naive lymphocytes,
inflammatory response, adhesion of immune cells, cell
movement, migration of cells, chemotaxis of cells, cell
movement of phagocytes, chemotaxis of monocytes, cell
movement of monocytes and fever;
ii) discarding the batch of the glatiramer acetate related
drug product as unacceptable for release if the level of
biological activity selected from the group consisting of
immune response to antigen presenting cells,
differentiation of effector lymphocytes, suppression of T
lymphocytes and activation of Foxp3 positive regulatory T
cells is decreased relative to a reference standard or if
the level of biological activity selected from the group
consisting of expansion of mononuclear leukocytes,
proliferation of T lymphocytes, expansion of lymphocytes,
differentiation of naive lymphocytes, inflammatory
response, adhesion of immune cells, cell movement,
migration of cells, chemotaxis of cells, cell movement of
phagocytes, chemotaxis of monocytes, cell movement of
monocytes and fever is increased relative to a reference
standard.
A method of identifying suboptimal activity of a glatiramer
acetate related drug substance or drug product comprising the
steps of:
a ) administering a glatiramer acetate related drug substance
or drug product to a rodent;
b ) determining the level of biological activity of the
rodent selected from the group consisting of, immune
response to antigen presenting cells, differentiation of
effector lymphocytes, suppression of T lymphocytes,
activation of Foxp3 positive regulatory T cells,
expansion of mononuclear leukocytes, proliferation of T
lymphocytes, expansion o f lymphocytes, differentiation o f
naive lymphocytes, inflammatory response, adhesion o f
immune cells, cell movement, migration of cells,
chemotaxis of cells, cell movement o f phagocytes,
chemotaxis of monocytes, cell movement of monocytes and
fever; and
c ) identifying the glatiramer acetate related drug substance
or drug product as causing a suboptimal activity if the
level of biological activity selected from the group
consisting of immune response to antigen presenting
cells, differentiation of effector lymphocytes,
suppression o f T lymphocytes and activation of Foxp3
positive regulatory T cells is decreased relative to a
reference standard or if the level o f biological activity
selected from the group consisting o f expansion of
mononuclear leukocytes, proliferation of T lymphocytes,
expansion of lymphocytes, differentiation of naive
lymphocytes, inflammatory response, adhesion of immune
cells, cell movement, migration o f cells, chemotaxis of
cells, cell movement o f phagocytes, chemotaxis of
monocytes, cell movement of monocytes and fever is
increased relative to a reference standard,
thereby identifying suboptimal activity o f a glatiramer
acetate related drug substance or drug product .
A method o f identifying suboptimal activity o f a glatiramer
acetate related drug substance or drug product comprising the
steps of:
a ) administering a glatiramer acetate related drug substance
or drug product to a rodent
b ) determining the level of expression in the rodent of one
or more genes selected from the group consisting of Ecml,
Presl, Pdlim4, Gpr83, Ifng, 1124, LOC100046608, Gm590,
Gprll4, Tmie, Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c, Als2cl,
2810410P22Rik, Arl5a, Gbp2, Rasgrpl, Ankrd37, Tpil,
4930583H14Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igf bp4 ,
Oas2, Bclllb, Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl,
Chi311 , Anxa3 , Hp, Lyz2 , Lyz , Ferll3 , Sirpa, Cd63 ,
Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp, S100a8,
S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3,
Gpr84, Acta2, Lcn2 , Hmoxl, Tpsabl, Ccl4, 112, Inhba,
Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2, Ilia,
Ccl3, 6720418B0lRik, 5830496LllRik, Cd8bl, Fcgrt,
LOC385615 and Scml4; determining the level of expression
of one or more genes selected from the group consisting
of the genes presented in Table 8 ; determining the level
of expression of one or more genes selected from the
group consisting of the genes presented in Table 10;
determining the level of expression of one or more genes
selected from the group consisting of FoxP3 , GPR83, CD14,
TLR2, IFNG, CD40 and IL1B; determining the level of
expression of one or more genes selected from the group
consisting of the genes presented in Table 12; or
determining gene set enrichment analysis for genes
associated with at least one cell type selected from the
group consisting of FoxP3+ CD4+ T cells, CD4+ T cells
CD8+ T cells, gamma delta T cells, natural killer T
cells, CD4+ CD8+ T cells, macrophage cells, monocyte
cells stromal cells, multi-lineage progenitor cells,
dendritic cells, fibroblastic reticular cells,
fibroblasts and granulocytes; and
identifying the glatiramer acetate related drug substance
or drug product as causing a suboptimal activity if the
level of expression of a gene selected from the group
consisting of Ecml, Presl, Pdlim4, Gpr83, Ifng, 1124,
LOC100046608, Gm590, Gprll4, Tmie, Rasgrpl, Myo6, Pfkp,
Uspl8, Arl4c, Als2cl, 2810410P22Rik, Arl5a, Gbp2,
Rasgrpl, Ankrd37, Tpil, 4930583Hl4Rik, Ifit3, LOC667370,
Klhdcl, Cd247, Igfbp4, Oas2 Bclllb, 6720418B0lRik,
5830496LllRik, Cd8bl, Fcgrt, LOC385615 and Scml4 is
decreased relative to a reference standard or if the
level of expression of a gene selected from the group
consisting of Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl,
Chi311, Anxa3 , Hp, Lyz2, Lyz, Ferll3, Sirpa, Cd63,
Clec4n, Clec4d, EG433016, Stfal, Chi313 Ngp, Sl00a8,
S100a9, Clecsf9, Saa3 , 5033414K04Rik, Slc7all, Slpi,
Cdl4, Fpr2, Fcgr3 , F10, Gpnmb, Tgfbi, Mmpl4, Slcllal, C3 ,
Gpr84, Acta2, Lcn2, Hmoxl, Tpsabl, Ccl4, 112, Inhba,
Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb, Cxcl2, Ilia,
and Ccl3, is increased relative to a reference standard;
identifying the glatiramer acetate related drug substance
or drug product as causing a suboptimal activity if the
level of expression of a gene selected from the group
consisting of the genes presented in Table 8 is not
substantially identical to the level of expression of a
reference standard; identifying the glatiramer acetate
related drug substance or drug product as causing a
suboptimal activity if the level of expression of a gene
selected from the group consisting of the genes presented
in Table 10 is not substantially identical to the level
of expression of a reference standard; identifying the
glatiramer acetate related drug substance or drug product
as causing a suboptimal activity if the level of
expression of a gene selected from the group consisting
of GPR83, IFNG and Foxp3 is decreased or if the level of
expression of a gene selected from the group consisting
of CD14, CD40, TLR2 and ILlB is increased; identifying
the glatiramer acetate related drug substance or drug
product as causing a suboptimal activity if the level of
expression of a gene selected from the group consisting
of the genes identified in Table 12 as FoxP3+ T cell
genes is decreased or if the level of expression of a
gene selected from the group consisting of the genes
identified in Table 12 as macrophage genes and the genes
identified in Table 12 as monocyte genes is increased; or
identifying the glatiramer acetate related drug substance
or drug product as causing a suboptimal activity if gene
set enrichment analysis indicates downregulation or a
lack of upregulation for genes associated with at least
one cell type selected from the group consisting of
FoxP3 + CD4+ T cells, CD4+ T cells CD8+ T cells, gamma
delta T cells, natural killer T cells and CD4+ CD8+ T
cells or if gene set enrichment analysis indicates
upregulation or a lack of downregulation for genes
associated with at least one cell type selected from the
group consisting of macrophage cells, monocyte cells
stromal cells, multi-lineage progenitor cells, dendritic
cells, fibroblastic reticular cells, fibroblasts and
granulocytes ,
thereby identifying suboptimal activity of the glatiramer
acetate related drug substance or drug product .
20. The method of any one of claims 18-19, wherein the level of
expression is determined in the blood.
21. The method of claim 20, wherein the level of expression is
determined in PBMCs .
22. The method of any one of claims 18-19, wherein the reference
standard is the level of expression prior to administration of
the glatiramer acetate related drug substance or drug product.
23. The method of any one of claims 18-19, wherein the reference
standard is the level of expression after administration of
glatiramer acetate drug substance or drug product.
24. The process of claim 4 , claim 18 or claim 19, wherein the
rodent is a mouse.
25. The process of claim 24, wherein the mouse is a female (SJL X
BALB/C) Fl mouse.
26. The process of claim 24 or claim 25, wherein the mouse is
about 8 to about 12 weeks old.
27. The process of claim 1 or claim 2 , wherein the primary culture
is a culture of spleen cells.
28. The process of claim 1 or claim 2 , wherein the primary culture
is a culture of lymph node cells.
29. The process of claim 28, wherein the primary culture of spleen
cells is prepared about 3 days after immunization.
30. The process of any one of claims 1-14, wherein the incubation
of step d ) is for about 24 hours.
31. The process or method of any one of claims 1-30, wherein the
glatiramer acetate related drug substance is a glatiramoid or
wherein the glatiramer acetate related drug product comprises
a glatiramoid.
32. The process or method of any one of claims 1-30, wherein the
glatiramer acetate related drug substance is a glatiramoid
other than glatiramer acetate drug substance or wherein the
glatiramer acetate related drug product comprises a
glatiramoid other than glatiramer acetate drug substance .
33. The process or method of any one of claims 1 , 12, 15 or 19
comprising the step of determining the level of expression of
at least one gene selected from the group consisting of Ecml,
Presl, Pdlim4, Gpr83 , Ifng, 1124, LOC100046608 , Gm590, Gprll4,
Tmie, Rasgrpl, Myo6, Pfkp, Uspl8, Arl4c, Als2cl,
2810410P22Rik, Arl5a, Gbp2 , Rasgrpl, Ankrd37, Tpil,
4930583Hl4Rik, Ifit3, LOC667370, Klhdcl, Cd247, Igfbp4, Oas2,
Bclllb, Fscnl, Ctsg, Mpo, Prtn3 , Lyzs, Emrl, Chi311, Anxa3 ,
Hp, Lyz2, Lyz, Ferll3 , Sirpa, Cd63, Clec4n, Clec4d, EG433016,
Stfal, Chi313 Ngp, Sl00a8, Sl00a9, Clecsf9, Saa3 ,
5033414K04Rik, Slc7all, Slpi, Cdl4, Fpr2 , Fcgr3 , F10, Gpnmb,
Tgfbi, Mmpl4, Slcllal, C3 , Gpr84, Acta2, Lcn2 , Hmoxl, Tpsabl,
Ccl4, 112, Inhba, Cxcll, Serpinb2, Uppl, Gprl09a, Gp38, Illb,
Cxcl2, Ilia, Ccl3, 6720418B0lRik, 5830496LllRik, Cd8bl , Fcgrt,
LOC385615 and Scml4.
34. The process of claim 1 comprising the step of determining the
level of expression of at least one gene selected from the
group consisting of Foxp3 , 112, Ilia, Illb, C3 , Sl00a8,
Sl00a9, Cxcl2, Cxcl3 , Ccl4, Ccl3 and Cdl4.
35. The process of claim 1 comprising the step of determining the
level of expression of at least one gene selected from the
group consisting of genes regulated by glatiramer acetate drug
substance or drug product in Gene Expression Omnibus accession
number GSE40566.
36. The process of claim 1 comprising the step of determining the
level of expression of at least one gene selected from the
group consisting of CD40, CD86, GATA3 , HLA-DMA, HLA-DMB, ICOS,
IFNG, IFNGR2, IL2 , IL13 , IL4, IL18, IL12RB1, IL17A, IL17F,
IL18R1, IL2RA, IL2RG, IL4R, IL6R, TBX21, TGFBR2 , TNF, F0XP3 ,
IL10RB, KLRDl, CD69, LTB, CD83 , PRFl, CAMK2D, LTA, FSCNl ,
TLR7, CSF2, CCR7, FASLG, ILIA, CCL5 , CD8B, CXCL10, TLR2 , CCL ,
TLR7, IGHA1, IL24, SOCS1, OAS1, JAKl , PTPN2 , IFITMl , IFI35,
STAT2, BCL2, MVD, FDPS, SQLE, NSDHL, DHCR24, Acat2/Acat3,
MSMOl, LSS, CYP51A1, NFKBIE, PIK3R1, PPP3CC, CD3D, IL2RB,
PTE , CD3G, ICOS, CAMK2D, NFAT5 , LAT, ITK, H2-M2, FASLG, LIF,
IGHA1, PRKACB, SGKl , MAPK11, TSC22D3, JU , FKBP5 , ADRB2 ,
MAP3K1, MAPK12, POU2F1, SMARCA2 , CDKNlA, TGFB3 , HSP90AA1,
DHCR24, CCR5, and CXCL9 .
37. The process or method of any one of claims 1 , 12, 15 or 19
comprising the step of determining the level of expression of
at least one gene selected from the group consisting of the
genes presented in Table 8 .
38. The process or method of any one of claims 1 , 12, 15 or 19
comprising the step of determining the level of expression of
at least one gene selected from the group consisting of the
genes presented in Table 10.
39. The process or method of any one of claims 1 , 12, 15 or 19
comprising the step of determining the level of expression of
at least one gene selected from the group consisting of FoxP3 ,
GPR83, CD14, TLR2 , IFNG, CD40 and ILlB.
40. The process or method of any one of claims 1 , 12, 15 or 19
comprising the step of determining the level of expression of
at least one gene selected from the group consisting of the
genes presented in Table 12 .
41. The process or method of any one of claims 1 , 12, 15 or 19
comprising the step of determining gene set enrichment
analysis for genes associated with at least one cell type
selected from the group consisting of FoxP3+ CD4+ T cells,
CD4+ T cells CD8+ T cells, gamma delta T cells, natural killer
T cells, CD4+ CD8+ T cells, macrophage cells, monocyte cells
stromal cells, multi-lineage progenitor cells, dendritic
cells, fibroblastic reticular cells, fibroblasts and
granulocytes .
42. The process or method of claim 41, wherein determining gene
set enrichment analysis comprises the step of evaluating the
level of expression of at least one gene selected from the
group consisting of genes present in one or more of the ran
list files presented in Table 11.
43. The process of any of claims 12-17 wherein the reference
standard is medium.