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EP3164710A2 - Caractérisation biologique d'un produit médicamenteux à l'acétate de glatiramère, à l'aide de cellules humaines et de mammifères - Google Patents

Caractérisation biologique d'un produit médicamenteux à l'acétate de glatiramère, à l'aide de cellules humaines et de mammifères

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Publication number
EP3164710A2
EP3164710A2 EP15814349.5A EP15814349A EP3164710A2 EP 3164710 A2 EP3164710 A2 EP 3164710A2 EP 15814349 A EP15814349 A EP 15814349A EP 3164710 A2 EP3164710 A2 EP 3164710A2
Authority
EP
European Patent Office
Prior art keywords
expression
level
glatiramer acetate
group
cells
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP15814349.5A
Other languages
German (de)
English (en)
Other versions
EP3164710A4 (fr
Inventor
Michael Hayden
Fadi George TOWFIC
Sarah Elisabeth Kolitz
Benjamin James ZESKIND
David Ladkani
Tal Hasson
Liat Hayardeny
Iris Grossman
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Teva Pharmaceutical Industries Ltd
Original Assignee
Teva Pharmaceutical Industries Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Teva Pharmaceutical Industries Ltd filed Critical Teva Pharmaceutical Industries Ltd
Publication of EP3164710A2 publication Critical patent/EP3164710A2/fr
Publication of EP3164710A4 publication Critical patent/EP3164710A4/fr
Withdrawn legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics

Definitions

  • MS Multiple sclerosis
  • CNS central nervous system
  • R relapsing-remitting
  • RRMS progressive course leading to neurologic deterioration and disability.
  • 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.
  • SP secondary progressive
  • PP primary progressive
  • 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.
  • 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.
  • IFNB cytokine interferon beta
  • 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 anti-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) .
  • APL altered peptide ligand
  • MBP myelin basic protein
  • 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.
  • APCs antigen presenting cells
  • TCR T cell receptor
  • 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 antigen- nonspecific modulation of APC function. Copaxone ® treatment promotes development of anti-inflammatory type II monocytes characterized by an increase in interleukin (IL)-10 and transforming growth factor- beta (TGF- ⁇ ) and decreased production of IL-12 and tumor necrosis factor (TNF) (32) .
  • IL interleukin
  • TGF- ⁇ transforming growth factor- beta
  • TNF tumor necrosis factor
  • 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) contacting mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; and c) i) determining the level of expression of at least one gene selected from the group consisting of ABCF2, ABI2, ACP6, AFG3L2,
  • CD44 CD9, CFP, C0L6A1, CRIP2, EPB41, Famll9a, FGR, FOX03B, HSD11B1, HSPD1P6, LOC387790, PEG1, YB, 0LIG1, PLD1, PPP4R2, PRDM1, RBM6, SNX27 , SOD2, STATH, TARP, TREM1, TRGC2, UBN2, and ZCCHC7 (hereinafter Gene Group 4); v) determining the level of expression of at least one gene selected from the group consisting of ADAM9, ADAMDEC1, AKR1C2, ANXA2, ANXA2P2, ARHGAP18, ARHGAP18, ARL6IP5, ARL6IP5 , ATP2C1, BID, BIRC3, BTG1, CARD15 , C10RF21, C130RF31, C50RF13, C50RF32, C9ORF130, CAST, CCL2 , CCL5, CD14, CD300A, CD36, CD
  • TREM1, TXNL2, VPS33A, and VSNL1 (hereinafter Gene Group 5); vi) determining the level of expression of at least one gene selected from the group consisting of ACTN4, BTBD14A, C14ORF10, CISH, CLK1, CRLF3 , FAM62A, FBX045, GAPDHS, HDAC4, HIC2, HNRPD, HSPD1, LOC648342, MYB, NAPB, OXCT2, SERPINB2, SFRS14, SPFH1,
  • STT3B, WDFY1, ZNF250, and ZNF566 (hereinafter Gene Group 6) ; vii) determining the level of expression of at least one gene selected from the group consisting of A2 , ABCB1, ABCC3, ABHD2, ACPP, ADAMDEC1, ADFP, ADORA2B, ADO A3 , AHNAK, ALCAM, AN H, ANKRD57, ANXA2, ANXA2P2, APBB1IP, AQP1, ARHGAP18 , ARHGAP20,
  • GLIS3 GPC1, GPR35, H2A/R, HAVCR2 , HMCN1, HIPK2, HIST2H2AA, HIVEP1, HMOX1, HSPB7 , ICAM1, ID2, ID2B, IFI30, IFI44, IFNGR1, IGFBP3, IL2RG, IL4I1, IL10RA, IL27RA, IL7R, IL10, INA, IRF7, ITGB5, ITGB7 , ⁇ 1505, KIAA1706, KMO, LBH, LFNG, LILRB1, LILRB2 , LMNA, LOC51334, LOC201895, LOC284262, LOC51334, LOC643424, LOC643834, LOC643847, LOC644242, LOC645238, LOC650429, LOC650446, LOC652543, LOC653610, LOC653754, LPAAT- THETA, LPXN, MAF, MAFB, MAML2,
  • NPTX1, PDCD6, and TIPARP (hereinafter Gene Group 9); x) determining the level of expression of at least one gene selected from the group consisting of ADRB2, COTL1, LOC285758, LOC644137, MALATl, PRG1, RNF43, SATl, THAP5, TIMP3, and TSC22D1 (hereinafter Gene Group 10); xi) determining the level of expression of at least one gene selected from the group consisting of AW011738, Bst2, Daxx, Gml6340, Hck, Herc6, Ifi202b, Ifi203, Ifi204, Ifi44, Ifi441, Ifit2, Inpp5b, LOC100044068, LOC100862473, Mxl, Oasll, Phflld, Oyhinl, Sdc3, Setdb2, Tor3a, Uspl8, and Zcchc2 (hereinafter Gene Group 11); xii) determining the level of expression of at least one gene selected from the group
  • IL4I1, MSC, NQOl, PPP1R15A, PRDM1, SLC7A11, SRXN1, TIPARP, T EM138 , TXNRD1 , and VEGF (hereinafter Gene Group 17); xviii) determining the level of expression of at least one gene selected from the group consisting of BCL2, CACNA2D3, C130RF18, C20ORF103, C50RF13, CDCA7, DEPDC6, GATM, HAL, HSPA1A, HSPC049,
  • LOC645919, LRMP, OAF, POU4F2, RASGRP2, RET, SERPINB2, SERPINB8, SPFH1, and TDRD7 (hereinafter Gene Group 18); xix) determining the level of expression of at least one gene selected from the group consisting of ABCC1, ABHD12, ABHD5, ACPP, ACSL1, ADFP, ADORA2B, ADORA3, AHRR, AKNA, AKR1C1, AKR1C2, A R1C3, ALAS1,
  • PRKCA PRKCA
  • PSCD4, PSCDBP PS D1, PTAFR, PTGS1, PTPN14, PTPRE, PTX3, QPRT, RAB13, RAB27B, RAB38 , RAB6IP1, RAI17, RAP2B, RAPGEF1 , RCN1, RELB, RGL1, RGS1, RGS2, RIN3, RIT1, RND3, RSNL2, RSP03, RUNX3, SAMSN1, SAP30, SASH1, SAT1, SDC4, SEMA4C, SERPINE2, SERTAD1, SFRS7, SGK, SH3GL1 , SH3TC1, SLAMF8, SLC15A3, SLC16A3,
  • TNFRSF10D TNFRSF1B, TNFRSF21 , TNFSF13B, TNFSF7, TP53BP2, TRAF3, TRAF3IP2, TRIB1, TRIB3 , TRIM16, TRIM16L, TRPA1, TRPS1, TRPS1, TSHZ3 , TTLL4, TXN D1, UBE2S , UGCG, ULBP2, UPP1, URP2, VASH1, VEGF, VSNL1, YRDC, ZBTB24, ZCCHC10, ZFAND5, ZFP36L1, ZNF366, ZNF516, and ZNF697 (hereinafter Gene Group 19) ; xx) determining the level of expression of at least one gene selected from the group consisting of ABHD14B, ACTN1, ACY1L2, ADA, ADD2 , AFF1 ,
  • Gene Group 23 determining the level of expression of at least one gene selected from the group consisting of IFNg, TNF, CCL3, CXCL8, and IL-10 (hereinafter Gene Group 24); xxv) determining the level of expression of at least one gene selected from the group consisting of MP9, CCL2,
  • CCL5, CXCL1, and ILIB (hereinafter Gene Group 25); xxvi) determining the level of expression of at least one gene selected from the group consisting of MMP9, CXCL10, CCL2, and CCL5 (hereinafter Gene Group 26) ; xxix) determining the level of expression of at least one gene selected from the group consisting of CCL3 , MMP9, CCL22, CCL24, CX3CL1, CCL20, CCL2 , TNF, IL8, CCL13, CCL5, IL1B, CCL8 , IL10, CXCL11, CXCL13, CXCL10, CCL7, CCL1, CXCL1, IFNg, CCL26, and MIF (hereinafter Gene Group 29) ; or xxx) determining the level of expression of at least one gene selected from the group consisting of CCL3, MMP9, CCL22,
  • the present invention also provides a process for discriminating between glatiramer acetate related drug substances or drug products comprising the steps of: a) characterizing two or more glatiramer acetate related drug substances or drug products according to the process of the claimed invention to obtain characteristics of each of the glatiramer acetate related drug substances or drug products; and b) comparing the characteristics of the glatiramer acetate related drug substances or drug products obtained in step a) , thereby discriminating between glatiramer acetate related drug substances or drug products.
  • 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) contacting mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; and c) xxii) determining the protein level expression of at least one protein selected from the group consisting of RANTES, IP-10, and MMP-9 (hereinafter Protein Group A) ; xxiii) determining the protein level expression of at least one protein selected from the group consisting of IL10 and MCP-1 (hereinafter Protein Group B) ; xxiv) determining the protein level expression of at least one protein selected from the group consisting of IFNg, TNFa, MIP-la, IL-8, and IL-10 (hereinafter Protein Group C) ; xxv) determining the protein level expression of at least one protein selected
  • 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) contacting mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; and c) i) determining the protein level expression of at least one protein produced by a gene selected from the group consisting of Gene Group 1; ii) determining the protein level expression of at least one protein produced by a gene selected from the group consisting of Gene Group 2, wherein if IL1B, IL10, or MMP9 is the at least one gene selected in part (c) (ii) , then selecting at least a second different gene from the group other than IL1B, IL10, or MMP9; iii) determining the protein level expression of at least one protein produced by a gene selected from the group consisting of Gene Group 3;
  • Figure 1 Framework of studies conducted in mouse splenocytes and human THP-1 monocyte cell line.
  • Figure 2 Roadmap for human THP-1 monocyte cell line study.
  • Figure 3 Levels of Copaxone ® measured over time in cell culture medium from human THP-1 monocyte cell line. Copaxone ® concentration in medium over time remains steady in the range of 44-52 g/mL over 24 hours.
  • FIGS. 4A-D Differentially expressed genes in human THP-1 monocyte cell line (a) Increased expression of IL10 with GA treatment at 6 hours for the single IL10 probeset on the array (207433_at) (FDR adjusted p ⁇ 3.1e-9); (b-d) Increased expression of IL1RN with GA treatment at 6 hours for multiple probesets (adjusted p values as shown) .
  • Figure 5 Pathways enriched in experimental systems in mouse splenocytes, human THP-1 monocyte cell line and PBMCs from MS patients among top genes modulated by Copaxone ® at 6 hours (subject to FC and adjusted p value filters of 1.5 and le-5, respectively).
  • the volcano plot show - log (adjusted p value) for the enrichment plotted versus the fold enrichment score from DAVID for each pathway. A selected subset of pathways are labeled.
  • Figure 6 Probeset for cytokine-cytokine receptor interaction pathway genes significantly modulated by Copaxone ® at 6 hours in human THP-1 monocyte cell line (subject to FC and adjusted p value filters of 1.5 and le-5, respectively) .
  • the volcano plot show - log (adjusted p value) for differential pexpression plotted versus the fold change from LIMMA for each probeset.
  • FIG. 7 MMP9 is significantly upregulated by Probioglat stimulation in human THP-1 monocyte cell line compared to Copaxone ® stimulation at 6 and 24 hours (FDR adjusted p values for each timepoint are 2.74e- 6, 0.098, and 0.004 for 6, 12, and 24 hours, respectively).
  • FIG. 8 CD14 expression in human THP-1 monocyte cell line is significantly higher with Probioglat stimulation compared to Copaxone ® stimulation at 6 hours.
  • Figure 9 Focus on the "response to LPS" pathway as differentially expressed by Probioglat versus Copaxone ® at 6 hours in human THP-1 monocyte cell line.
  • Figure 10 Pathway-level analysis depicts enrichment among genes upregulated by Probioglat stimulation compared with Copaxone ® at 6 hours in human THP-1 monocyte cell line
  • the volcano plot shows -log (adjusted p value) for the enrichment plotted versus the fold enrichment score from DAVID for each pathway;
  • the volcano plot shows -log (adjusted p value) for differential expression plotted versus the fold change from LIMMA for each probeset. A selected subset of pathways are labeled.
  • FIG. 11 Each present probeset for ICAM1 is significantly upregulated by Probioglat stimulation compared to Copaxone ® stimulation at 6 hours in human THP-1 monocyte cell line.
  • FIG. 12 CISH is downregulated by Probioglat stimulation compared to Copaxone ® stimulation at 6 hours in human human THP-1 monocyte cell line .
  • Figure 13 Upregulation of IL10 with GA treatment in all three studies in mouse splenocytes, human THP-1 monocyte cell line and PBMCs from MS patients.
  • FIGS. 15A-B Higher levels of (a) CD14 and (b) CD40 (bottom) are induced by a Probioglat generic than by Copaxone ® or other generics in human monocytes .
  • Figure 16 Expression of IL1B in Copaxone ® and multiple generics in mouse splenocytes .
  • Figure 17 Expression of CD44 in Copaxone ® and Escadra generic in human monocytes .
  • Figure 18 IL27 expression in Copaxone ® and various generics in mouse splenocytes .
  • Figure 19 MP9 expression induced by Copaxone ® , Probioglat, and other generics in human monocytes.
  • Figure 20 Significant gene expression difference are observed between Copaxone ® and the purported generics Natco and Escadra in many genes, including several relevant to MS .
  • FIG. 21 MMP14 expression is significantly elevated by TV-5010 relative to Copaxone ® in mouse splenocytes.
  • FIG. 22 The expression of PGRMC1 and IL1B shows significantly different expression following stimulation with proposed generics from different manufacturers in human THP-1 monocytes.
  • Figure 23 Pathway-level analysis depicts enrichment among genes upregulated by Copaxone ® stimulation compared with mannitol control at 6 hours in human THP-1 monocyte cell line.
  • Figure 24 IL1RN is significantly upregulated by Copaxone ® stimulation in human THP-1 monocyte cell line compared to mannitol control at 6 hours (FDR adjusted p value ⁇ 2.8e-10).
  • Fig. 25a Higher level of CYP1B1 is induced by Polimunol generic than by Copaxone ® in human THP-1 monocyte cell line at 6 hours (FDR adjusted p values ⁇ 1.5e-10).
  • Fig. 25b Higher level of GPR68 is induced by Polimunol generic than by Copaxone ® in human THP-1 monocyte cell line at 6 hours (FDR adjusted p values ⁇ 1.5e-5).
  • Figure 26 Lower level of ADRB2 is induced by Polimunol generic than by Copaxone ® in human THP-1 monocyte cell line at 6 hours (FDR adjusted p value ⁇ 0.009) .
  • FIGS. 27A-E Copaxone ® treatment in mouse splenocytes upregulated (a- b) anti-inflammatory cytokines and (c-d) markers of regulatory T cells, and (e) downregulated pro-inflammatory cytokines.
  • Figure 28 Pathways enriched among probesets modulated by Copaxone ® relative to medium in splenocytes from mice immunized with Copaxone ® .
  • Figure 29 IL18 expression is reduced to a greater extent by Copaxone ® than Polimunol, regardless of which substance is utilized for the immunization in mouse splenocytes.
  • Figure 30 Pathways enriched among top probesets upregulated by Polimunol relative to Copaxone ® (in Copaxone ® -immunized mice) .
  • Figure 31 Pathways enriched among top probesets upregulated by Polimunol relative to Copaxone ® (in Polimunol-immunized mice) .
  • Fig. 32a IL-18 signaling pathway (from Dinarello, Front Immunol, 2013; Figure 1) .
  • Fig. 32b Mice definicient for IL-18 are resistant to EAE (from Shi, J. Immuno, 2000; Figure la) .
  • Figure 33 Type I interferon signaling pathway (from Trinchieri et al, Journal of Experimental Medicine, 2010; Figure 1)
  • FIGS. 34A-C Anti-inflammatory gene IL1RN is signfiicantly upregulated by Copaxone ® in human THP-1 monocyte cell line.
  • Figure 35 Pathways enriched among top probesets modulated by Copaxone ® relative to mannitol control in human THP-1 monocyte cell line .
  • Figure 36 Genes in the cytokine-cytokine receptor interaction pathway modulated by Copaxone ® relative to mannitol control (each point represents a probeset, labeled with its gene annotation) in human THP- 1 monocyte cell line.
  • CYP1B1 is significantly upregulated by Polimunol relative to Copaxone ® .
  • Figure 38 Pathways significantly enriched among top probesets differentially expressed between Polimunol and Copaxone ® .
  • Figure 39 Genes in the cytokine-cytokine receptor interaction pathway, which is significantly enriched among top probesets differentially expressed between Polimunol and Copaxone ® (each point represents a probeset, labeled with its gene annotation) .
  • Fig. 40a Volcano plot showing probesets driving the enrichment of the response to lipopolysaccharide pathway among probesets upregulated by Polimunol relative to Copaxone ® .
  • the dashed line indicates significance level of adjusted p value ⁇ 0.05.
  • the dashed line indicates significance level of adjusted p value ⁇ 0.05.
  • Figure 41 Expression levels of genes differing between Probioglat and GA
  • MMP9 is significantly upregulated following stimulation by Probioglat compared to GA at 6 and 24 hours (FDR-adjusted p values for the single MMP9 probeset on the chip, 203936_s_at, are 2.74e-6, 0.098, and 0.004 for the 6, 12, and 24 hour timepoints, respectively);
  • CD14 expression is significantly higher with stimulation by Probioglat compared to GA at 6 hours (the single CD14 probeset on the chip is shown, 201743_at) ;
  • Both present ICAM1 probesets are significantly upregulated following stimulation by Probioglat compared to GA at 6 hours (A: probeset 202637_s_at; B: probeset 202638_s_at) ;
  • CISH is downregulated following stimulation by Probioglat compared to GA at 6 hours (both present probesets are shown, A: probeset 223961_s_at; B: probeset 223377_x_at)
  • Figure 42 Expression levels of genes differing between Probioglat and GA by qRT-PCR in primary human monocytes.
  • CCL2 (p ⁇ 0.009)
  • CCL5 (p ⁇ 0.029)
  • CXCL10 (p ⁇ 0.020)
  • MMP9 (p ⁇ 0.009)
  • IL1RN (p ⁇ 0.013) are expressed more highly under Probioglat stimulation relative to GA stimulation.
  • PCA Principal Component Analysis
  • FIGS. 45A-B Protein levels at 24 hours are consistent with upregulation of pro-inflammatory mRNA markers by Probioglat vesus Copaxone ® treatment at 6h
  • CCL, CXCL10 and MMP-9 protein levels are higher with Probioglat versus Copaxone ® at 24 hr, consistent with mRNA level observations by RT-PCR and microarray in THP-1 cells
  • CCL2 and IL-10 protein levels are higher with Probioglat versus Copaxone ® at 24 hr, consistent with mRNA level observations by microarray (not tested by RT-PCR in THP-1 cells.
  • FIGS. 47A_E Levels of Secreted Protein with Polimunol Versus GA Treatment: Levels of IFNg, TNFa, MlPla (CCL3) , IL-8 (CXCL8 ) , and IL- 10 were higher with Polimunol versus GA treatment.
  • FIGS. 48A-E Levels of Secreted Protein with Polimunol Versus GA Treatment: Consistent with gene level data, levels of MMP-9, MCP-1 (CCL2), RANTES (CCL5), Gro-a (CXCL1) , and IL-lb were higher with Polimunol versus GA treatment.
  • FIGS. 49A-E Levels of Secreted Protein with Probioglat Versus GA Treatment: Levels of IFNg, TNFa, MlPla (CCL3) , IL-8 (CXCL8), and IL- 10 were higher with Probioglat versus GA treatment.
  • FIGS. 50A-D Levels of Secreted Protein with Probioglat Versus GA Treatment: Consistent with gene level data, levels of MMP-9, MCP-1 (CCL2), RANTES (CCL5), and IP-10 (CXCL10) were higher with Probioglat versus GA treatment.
  • 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) contacting mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; and c) i) determining the level of expression of at least one gene selected from the group consisting of Gene Group 1; ii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 2, wherein if IL1B, IL10, or MMP9 is the at least one gene selected in part (c) (ii) , then selecting at least a second different gene from the group other than XL1B / IL10 / or P9; iii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 3; iv) determining the level of expression of at least one gene
  • step (c) comprises i) determining the level of expression of at least one gene selected from the group consisting of Gene Group 1; ii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 3; or vi) determining the level of expression of at least one gene selected from the group consisting of Gene Group 6.
  • all genes Gene Group 1 are selected for determining the level of expression.
  • the process comprises the step of selecting at least a second different gene from the group of (c) (i) and (c) (ii) other than IL10.
  • the process comprises the step of selecting at least a second different gene from the group of (c) (i) and (c) (ii) other than CARD15, CCL2, CCL5, CD14, IL10, THBD, or NFKBIA.
  • step (c) if two or more genes are selected in step (c) , then the second or additional gene selected is different from the other selected gene or genes
  • the level of expression is determined for all genes identified in Table 5 or Table 12 to be involved in one or more than one pathway.
  • the level of expression is determined for at least one gene identified in Table 5 or Table 12 to be involved in at least one pathway.
  • the level of expression is determined for at least one gene identified in Table 5 or Table 12 to be involved in two or more pathways. In one or more embodiments of the present invention, the level of expression is determined for at least one gene identified in Table 5 or Table 12 to be involved in three or more pathways.
  • the level of expression is determined for at least one gene identified in Table 5 or Table 12 to be involved in four or more pathways.
  • the level of expression is determined for at least one gene identified in Table 5 or Table 12 to be involved in five or more pathways.
  • the level of expression is determined for at least one gene identified in Table 5 or Table 12 to be involved in six pathways .
  • the level of expression is determined for at least one gene which is involved in only one pathway set forth in Table 5 or Table 12. In one or more embodiments of the present invention, the level of expression is determined for at least two genes identified in Table 5 or Table 12 to be involved in the same pathway.
  • the level of expression is determined for at least three genes identified in Table 5 or Table 12 to be involved in the same pathway.
  • the level of expression is determined for at least four genes identified in Table 5 or Table 12 to be involved in the same pathway.
  • the level of expression is determined for at least five genes identified in Table 5 or Table 12 to be involved in the same pathway.
  • the level of expression is determined for at least six genes identified in Table 5 or Table 12 to be involved in the same pathway. In one or more embodiments of the present invention, the level of expression is determined for all genes identified in Table 6 to be involved in one or more than one pathway.
  • the level of expression is determined for at least one gene identified in Table 6 to be involved in at least one pathway.
  • the level of expression is determined for at least one gene identified in Table 6 to be involved in two or more pathways.
  • the level of expression is determined for at least one gene identified in Table 6 to be involved in three or more pathways .
  • the level of expression is determined for at least one gene identified in Table 6 to be involved in four or more pathways . In one or more embodiments of the present invention, the level of expression is determined for at least one gene identified in Table 6 to be involved in five or more pathways .
  • the level of expression is determined for at least one gene identified in Table 6 to be involved in six or more pathways.
  • the level of expression is determined for at least one gene which is involved in only one pathway set forth in Table 6.
  • the level of expression is determined for at least two genes identified in Table 6 W.
  • the level of expression is determined for at least three genes identified in Table 6 to be involved in the same pathway. In one or more embodiments of the present invention, the level of expression is determined for at least four genes identified in Table 6 to be involved in the same pathway.
  • the level of expression is determined for at least five genes identified in Table 6 to be involved in the same pathway.
  • the level of expression is determined for at least six genes identified in Table 6 to be involved in the same pathway.
  • contacting the mammalian cells in step (b) comprises i) administering to a mammal a predetermined amount of glatiramer acetate related drug substance or drug product of step (a) , or ii) incubating the cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) , or a combination thereof; and wherein step (c) comprises i) determining the level of expression of at least one gene selected from the group consisting of Gene Group 1, thereby characterizing the glatiramer acetate related drug substance or drug product of step (a) .
  • contacting the mammalian cells in step (b) comprises i) administering to a mammal a predetermined amount of glatiramer acetate related drug substance or drug product of step (a), and ii) obtaining cells from the mammal at one or more predetermined time points; and wherein step (c) comprises determining the level of expression of at least one gene selected from the group consisting of Gene Group 1, thereby characterizing the glatiramer acetate related drug substance or drug product of step (a) .
  • the mammal is human and the cells are peripheral mononuclear blood cells.
  • the predetermined time point is 0, 1, 2, or 3 months.
  • contacting the mammalian cells in step (b) comprises incubating monocytic cell line cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) ; and wherein step (c) comprises determining the level of expression of at least one gene selected from the group consisting of Gene Group 1; ii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 2; iii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 3; iv) determining the level of expression of at least one gene selected from the group consisting of Gene Group 4; v) determining the level of expression of at least one gene selected from the group consisting of Gene Group 5; vi) determining the level of expression of at least one gene selected from the group consisting of Gene Group 6; vii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 7; viii) determining the level of expression of at least one gene
  • contacting the mammalian cells in step (b) comprises incubating monocytic cell line cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) ; and wherein step (c) comprises i) determining the level of expression of at least one gene selected from the group consisting of Gene Group 2; ii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 3; iii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 4; iv) determining the level of expression of at least one gene selected from the group consisting of Gene Group 5; v) determining the level of expression of at least one gene selected from the group consisting of Gene Group 6; vi) determining the level of expression of at least one gene selected from the group consisting of Gene Group 7; or vii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 8; ix) determining the level of expression of at
  • the mammalian cells are THP-1 cells.
  • contacting the mammalian cells in step (b) comprises i) immunizing a mammal with a predetermined amount of glatiramer acetate related drug substance or drug product, ii) preparing a culture of cells from the mammal of step i) at one or more predetermined time points after immunization, and iii) incubating cells from the culture of cells obtained from the mammal with an amount of the glatiramer acetate related drug substance or drug product of step (a) ; and wherein step (c) comprises i) determining the level of expression of at least one gene selected from the group consisting of Gene Group 1; xi) determining the level of expression of at least one gene selected from the group consisting of Gene Group 11; xii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 12; xiii) determining the level of expression of at least one gene selected from the group consisting of Gene Group 13;
  • the glatiramer acetate related drug substance or drug product of step (iii) is the same glatiramer acetate related drug substance or drug product of step (i) . In one or more embodiments of the present invention, the glatiramer acetate related drug substance or drug product of step (iii) is a different glatiramer acetate related drug substance or drug product of step (i) .
  • the incubation is for about 24 hours, for about 12 hours, or for about 6 hours.
  • the predetermined time point after immunization is 3 days.
  • the contacting of step (b) is in a cell culture.
  • the culture is a primary culture.
  • step (b) the contacting of step (b) is in a mammal.
  • the mammal is a rodent or human.
  • the glatiramer acetate related drug substance or drug product is other than glatiramer acetate drug substance or drug product.
  • the cell is of a type i) selected from the group of cell types consisting of FoxP3+ T cells, regulatory T cells, natural killer T cells, T helper 2 cells, CD8+ T cells, CD4+ T cells, B cells, macrophage cells, monocyte cells, eosinophils, dendritic cells, granulocytes, megakaryocytes, and myeloid progenitors; ii) selected from the group of cell types identified in Table 9; iii) selected from the group of cell types identified in Table 10; or iv) selected from the group of cell types identified in Table 11.
  • ii) selected from the group of cell types consisting of FoxP3+ T cells, regulatory T cells, natural killer T cells, T helper 2 cells, CD8+ T cells, CD4+ T cells, B cells, macrophage cells, monocyte cells, eosinophils, dendritic cells, granulocytes, megakaryocytes, and myeloid progenitors ii) selected from the group
  • the process of characterizing two or more glatiramer acetate related drug substances or drug products further comprises obtaining characteristics of each of the glatiramer acetate related drug substances or drug products; and comparing the characteristics of the drug related substances or drug products, thereby discriminating between glatiramer acetate related drug substances or drug products .
  • the mammal is a rodent or human.
  • the level of expression is determined in hematological cells.
  • the level of expression is determined in splenocytes .
  • the level of expression is determined in monocytes.
  • the monocytes are THP-1.
  • the level of expression is determined in peripheral blood mononuclear cells.
  • the peripheral blood mononuclear cells are from a human.
  • the human has previously been treated with a glatiramer acetate related drug substance or drug product.
  • the human is a naive human.
  • the human is a glatiramoid naive human. In one or more embodiments of the present invention, the human is afflicted with RRMS.
  • the rodent is a mouse .
  • the mouse is a female (SJL X BALB/C) Fl mouse.
  • the mouse is about 8 to 12 weeks old.
  • the primary culture is a culture of spleen cells.
  • the primary culture is a culture of lymph node cells.
  • the primary culture of spleen cells is prepared about 3 days after immunization.
  • the glatiramer acetate related drug substance is a glatiramoid or the glatiramer acetate related drug product comprises a glatiramoid.
  • the glatiramer acetate related drug substance is a glatiramoid other than glatiramer acetate related drug substance, or the glatiramer acetate related drug product comprises a glatiramoid other than glatiramer acetate drug substance .
  • 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) contacting a first group of mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; c) contacting a second group of mammalian cells of the same type with an amount of a reference standard; and d) determining a set of genes differentially expressed by the first group after the contacting of step (b) relative to genes expressed by the second group after the contacting of step (c) , thereby characterizing the glatiramer acetate related drug substance or drug product of step a) .
  • the cells are THP-1 cells.
  • the reference standard is glatiramer acetate related drug substance or drug product.
  • the reference standard is mannitol
  • the reference standard is medium.
  • the determining step (d) comprises comparing the expression of genes expressed by the first group to the expression of genes expressed by the second group.
  • the determining step (d) comprises comparing the expression of genes by bother the first group of cells and by the second group of cells to expression of the genes by the same type of cells exposed to mannitol or medium.
  • process for discriminating between glatiramer acetate related drug substances or drug products comprising the step of characterizing two or more glatiramer acetate related drug substances or drug products to obtain characteristics of each of the glatiramer acetate related drug substances or drug products; and comparing the characteristics of the glatiramer acetate related drug substances or drug products, thereby discriminating between glatiramer acetate related drug substances or drug products .
  • the improvement comprising including in the array of testing the steps of: a) characterizing the glatiramer acetate related drug substance according to the process of any one of the above characterization methods; and b) i) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is not substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions; ii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions;
  • the level of expression of one or more genes selected from the group consisting of Gene Group 11 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 12 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xiii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 13 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xiv) including the batch of the glatiramer acetate related drug substance in the
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is not substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of ABCF2, ABI2, ACP6, AFG3L2, ALMS1 , ARPC4, CALM3 , CCDC64, CD84, CDC6, CHAF1A, CLU, COX11, DLGAP1, DTX4, FAM49B, FHL1, FNTB, GYPC, HFE, LPHN1, OLAH, PATZ1, PDK1, POLI, REEP5 , RPS6KA2, SEC31A, SETBP1, SNRPA1, SYNCRIP, TNFSF9, TOMM40, TPM1, TSPAN13, UBAP2 , VAV3 , VDAC2 ,
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 3 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions .
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 4 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 5 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 6 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises i) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of ABI2, ARPC4, CD84, CLU, HFE, and IL10 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions; or ii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of ABCF2, ACP6, AFG3L2, CHAF1A, COX11, LPHN1, NACA, OLAH, POLI, SEC31A, SNRPA1, SYNCRIP, TNFSF
  • the characterizing the glatiramer acetate related drug substance further comprises, if, one or more genes selected from the group consisting of ABI2, ARPC4, HFE, and IL10 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions, then including the batch of the glatiramer acetate related drug substance in the production of the drug product.
  • the characterizing the glatiramer acetate related drug substance further comprises, if, one or more genes selected from the group consisting of ACP6, LPHN1, POLI, SEC31A, SYNCRIP, and TSHZ1 is downregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions, then including the batch of the glatiramer acetate related drug substance in the production of the drug product.
  • the characterizing the glatiramer acetate related drug substance further comprises i) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of CCL2, CCL5, M P1, MP9, CXCL10, CARD15 , CD14, ICAM1, BIRC3, THBD, NF BIA, IL10, PRDM1 is substantially identical to the level of expression by the same type of cells in the presence of glatiramer acetate drug substance under the same conditions; ii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of CISH and HSPD1 is substantially identical to the level of expression by the same type of cells in the presence of
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 5 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions .
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 6 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 7, is upregulated to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 8, is downregulated to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions.
  • a process for releasing a drug product comprising a glatiramer acetate related drug substance which process involves an array of testing, the improvement comprising including in the array of testing the steps of: a) characterizing the glatiramer acetate related drug product according to the process of any one of the above characterization methods; and b) i) releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is not substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug product under the same conditions; ii) releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions; iii) releasing the batch of
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is not substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug product under the same conditions.
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of ABCF2, ABI2, ACP6, AFG3L2, ALMS1 , ARPC4, CALM3, CCDC64 , CD84, CDC6, CHAF1A, CLU, COX11, DLGAP1, DTX4 , FAM49B, FHL1, FNTB, GYPC, HFE, LPHN1 , OLAH, PATZ1, PDK1, POLI, REEP5, RPS6KA2, SEC31A, SETBP1, SNRPA1, SYNCRIP, TNFSF9, TOMM40, TPM1, TSPA 13, UBAP2, VAV3, VDAC2 , and ZFAND6 is not substantially identical to
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 2 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions.
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 3 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions .
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 4 is substantially identical to the level of W 201
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 5, is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions.
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 6 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions.
  • the characterizing the glatiramer acetate related drug substance further comprises i) releasing the batch of the glatiramer acetate related drug product if the level of expression of one of more genes selected from the group consisting of ABI2, ARPC4, CD84, CLU, HFE, and IL10 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug product under the same conditions; or ii) releasing the batch of the glatiramer acetate related drug product if the level of expression of one of more genes selected from the group consisting of ABCF2, ACP6, AFG3L2, CHAF1A, COX11, LPHN1, NACA, OLAH, POLI, SEC31A, SNRPA1, SYNCRIP, TNFSF9, TOM 40, TSHZ1, T
  • the characterizing the glatiramer acetate related drug substance further comprises, if, one or more genes selected from the group consisting of ABI2, ARPC4, HFE, and IL10 is upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug product under the same conditions, then releasing the batch of the glatiramer acetate related drug product.
  • the characterizing the glatiramer acetate related drug product further comprises, if, one or more genes selected from the group consisting of ACP6, LPHN1, POLI, SEC31A, SYNCRIP, and TSHZ1 is downregulated relative to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions, then releasing the batch of the glatiramer acetate related drug product.
  • the characterizing the glatiramer acetate related drug product further comprises i) releasing the batch of the glatiramer acetate related drug product if the level of expression of one of more genes selected from the group consisting of CCL2, CCL5 , MMP1, MMP9, CXCL10, CARD15 , CD14, ICAM1, BIRC3, THBD, NFKBIA, IL10, PRDM1 is substantially identical to the level of expression by the same type of cells in the presence of glatiramer acetate drug substance under the same conditions; ii) releasing the batch of the glatiramer acetate related drug product if the level of expression of one of more genes selected from the group consisting of CISH and HSPD1 is substantially identical to the level of expression by the same type of cells in the presence of glatiramer acetate
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 5, is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions.
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 6 is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions.
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 7, is upregulated to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions.
  • the characterizing the glatiramer acetate related drug product further comprises releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 8, is downregulated to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions.
  • a glatiramer acetate related drug product produced by a process of the present invention, wherein the glatiramer acetate related drug product is other than glatiramer acetate drug product.
  • a glatiramer acetate related drug product other than glatiramer acetate drug product which is capable of inducing a level of expression of ABCF2, ABI2, ACP6, AFG3L2, ALMS1 , ARPC4, CALM3, CCDC64, CD84, CDC6, CHAF1A, CLU, COX11, DLGAP1, DTX4, FAM49B, FHL1, FNTB, GYPC, HFE, LPHN1, OLAH, PATZ1, PDK1, POLI, REEP5, RPS6KA2, SEC31A, SETBP1, SNRPA1, SYNCRIP, TNFSF9, TOMM40, TPM1, TSPAN13, UBAP2, VAV3, VDAC2, and ZFAND6 that is not substantially identical to the level of expression of the genes induced in the same tpe of cells and under the same conditions in the absence of the glatiramer acetate related drug product.
  • a glatiramer acetate related drug product wherein the glatiramer acetate related drug product is capable of inducing a level of expression of Gene Group 1.
  • the glatiramer acetate related drug product which is capable of upregulating genes ABI2, ARPC4, HFE, and IL10 relative to the level of expression of the genes in the same type of cells and under the same conditions in the absence of the glatiramer acetate related drug product, and capable of downregulating genes ACP6, LPHN1, POLI, SEC31A, SYNCRIP, and TSHZ1 relative to the level of expression of the genes in the same type of cells and under the same conditions in the absence of the glatiramer acetate related drug product.
  • a glatiramer acetate related drug product other than glatiramer acetate drug product which is capable of inducing a level of expression of Gene Group 7 and Gene Group 8, that is not substantially identical to the level of expression of the genes induced in the same type of cells and under the same conditions in the absence of the glatiramer acetate related drug product.
  • the glatiramer acetate related drug product which is capable of upregulating genes Gene Group 7, relative to the level of expression of the genes in the same type of cells and under the same conditions in the absence of the glatiramer acetate related drug product, and capable of downregulating genes Gene Group 8 relative to the level of expression of the genes in the same type of cells and under the same conditions in the absence of the glatiramer acetate related drug product
  • the process further comprises i) determining the one or more proteins produced by each of the one of more genes selected in step c) ; and ii) determining protein level expression for each protein in step i) .
  • the process further comprises i) determining the one or more proteins produced by each of the one of more genes selected in step b) ; and ii) determining protein level expression for each protein in step i) .
  • the process further comprises determining the set of proteins produced by each gene of the set of genes in step d) .
  • 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) contacting mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; and c) xxii) determining the protein level expression of at least one protein selected from the group consisting of Protein Group A; xxiii) determining the protein level expression of at least one protein selected from the group consisting of Protein Group B; xxiv) determining the protein level expression of at least one protein selected from the group consisting of Protein Group C; xxv) determining the protein level expression of at least one protein selected from the group consisting of Protein Group D; xxvi) determining the protein level expression of at least one protein selected from the group consisting of Protein Group E; xxvii) determining the protein level expression of at least one protein selected from the
  • 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) contacting mammalian cells with an amount of the glatiramer acetate related drug substance or drug product of step a) ; and c) i) determining the protein level expression of at least one protein produced by a gene selected from the group consisting of Gene Group 1; ii) determining the protein level expression of at least one protein produced by a gene selected from the group consisting of Gene Group 2, wherein if IL1B, IL10, or MMP9 is the at least one gene selected in part (c) (ii) , then selecting at least a second different gene from the group other than IL1B, IL10, or MMP9; iii) determining the protein level expression of at least one protein produced by a gene selected from the group consisting of Gene
  • step (b) comprises incubating monocytic cell line cells with an amount of the glatiramer acetate related drug substance or drug product of step (a) and wherein step (c) comprises xxii) determining the protein level expression of at least one protein selected from the group consisting of Protein Group A; xxiii) determining the protein level expression of at least one protein selected from the group consisting of Protein Group B; xxiv) determining the protein level expression of at least one protein selected from the group consisting of Protein Group C; xxv) determining the protein level expression of at least one protein selected from the group consisting of Protein Group D; xxvi) determining the protein level expression of at least one protein selected from the group consisting of Protein Group E; xxvii) determining the protein level expression of at least one protein selected from the group consisting of Protein Group F; or xxviii) determining the protein level expression of at least one protein selected from the group consisting of Protein Group
  • the process wherein the mammalian cells are THP-1 cells.
  • the process, wherein the incubation is for about 24 hours.
  • the process, wherein contacting the mammalian cells in step (b) comprises i) immunizing a mammal with a predetermined amount of glatiramer acetate related drug substance or drug product, ii) preparing a culture of cells from the mammal of step i) at one or more predetermined time points after immunization, and iii) incubating cells from the culture of cells obtained from the mammal with an amount of the glatiramer acetate related drug substance or drug product of step (a) , thereby characterizing the glatiramer acetate related drug substance or drug product of step (a) .
  • the process, wherein the glatiramer acetate related drug substance or drug product of step (iii) is the same glatiramer acetate related drug substance or drug product of step (i) .
  • step (iii) is a different glatiramer acetate related drug substance or drug product of step (i) .
  • the improvement comprising including in the array of testing the steps of: a) characterizing the glatiramer acetate related drug substance according to the process of any one of the above characterization methods; and b) xxii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more proteins selected from the group consisting of Protein Group A is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drug substance under the same conditions; xxiii) including the batch of the glatiramer acetate related drug substance in the production of the drug product if the level of expression of one or more proteins selected from the group consisting of Protein Group B is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drug substance under
  • a process for releasing a drug product comprising a glatiramer acetate related drug substance which process involves an array of testing, the improvement comprising including in the array of testing the steps of: a) characterizing the glatiramer acetate related drug product according to the process of any one of the above characterization methods; and xxii) releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more proteins selected from the group consisting of Protein Group A is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drug product under the same conditions; xxiii) releasing the batch of the glatiramer acetate related drug product if the level of expression of one or more proteins selected from the group consisting of Protein Group B is substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate related drug product under the same conditions; xxiv) releasing the steps of: a) characterizing the
  • a process for discriminating between glatiramer acetate related drug substances or drug products comprising the steps of: a) characterizing one or more glatiramer acetate related drug substances or drug products according to the process of any one of the above characterization methods to obtain characteristics of each of the glatiramer acetate related drug substances or drug products; b) obtaining characteristics of a glatiramer acetate drug substance or drug product; and c) comparing the characteristics of the glatiramer acetate related drug substances or drug products obtained in steps a) and b) , thereby discriminating between glatiramer acetate related drug substances or drug products.
  • a process for producing a drug product comprising a glatiramer acetate related drug substance comprising the steps of: a) characterizing the glatiramer acetate related drug substance according to the process of any one of the above characterization methods; and b) i) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is substantially identical to the level of expression by the same type of cells in the absence
  • Group 25 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xxvi) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 26 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xxix) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes selected from the group consisting of Gene Group 29 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; or xxx) discarding the batch of the glatiramer acetate related drug substance as unacceptable for inclusion in the drug product if the level of expression of one or more genes selected from the group consisting
  • a process for releasing a drug product comprising a glatiramer acetate related drug substance
  • the improvement comprising the steps of: a) characterizing the glatiramer acetate related drug product according to the process of any one of the above characterization methods; and b) i) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug product under the same conditions; ii) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene
  • Group 2 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions; iii) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 3 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions; iv) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 4 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions; v) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 5, is not substantially identical to the level of expression
  • Group 9 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions; x) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 10 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug product under the same conditions; xi) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 11 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xii) discarding the batch of the glatiramer acetate related drug product as unacceptable for release if the level of expression of one or more genes selected from the group consisting of Gene Group 12 is not substantially identical to the level of expression
  • a process for 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 mammal; b) characterizing the glatiramer acetate related drug substance or drug product according to the process of any one of the above characterization methods; and i) identifying the glatiramer acetate related drug substance or drug product as causing a suboptimal activity if the level of expression of one or more genes selected from the group consisting of Gene Group 1 is substantially identical to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance or drug product under the same conditions; ii) identifying the glatiramer acetate related drug substance or drug product as causing a suboptimal activity if the level of expression of one or more genes selected from the group consisting of Gene Group 2 is not substantially identical to the level of
  • 21 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xxii) identifying the glatiramer acetate related drug substance or drug product as causing a suboptimal activity if the level of expression of one or more genes selected from the group consisting of Gene Group
  • 26 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; xxix) identifying the glatiramer acetate related drug substance or drug product as causing a suboptimal activity if the level of expression of one or more genes selected from the group consisting of Gene Group
  • 29 is not substantially identical to the level of expression by the same type of cells in the presence of the glatiramer acetate drug substance under the same conditions; or xxx) identifying the glatiramer acetate related drug substance or drug product as causing a suboptimal activity if the level of expression of one or more genes selected from the group consisting of Gene Group
  • a process for producing a drug product comprising a glatiramer acetate related drug substance where the batch of the glatiramer acetate related drug substance is included in the drug product if the level of expression of one or more genes selected from one or more Gene Groups is substantially upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions, then conversely the batch of the glatiramer acetate related drug substance is discarded if the level of expression of one or more genes selected from one or more Gene Groups is not substantially upregulated relative to the level of expression by the same type of cells in the absence of the glatiramer acetate related drug substance under the same conditions as unacceptable for inclusion in the drug product.
  • a "naive human” is a human that has not been treated with any multiple sclerosis drug.
  • glatiramoid naive human is a human that has not been treated with any glatiramoid drug.
  • a glatiramoid naive human could have been treated with another multiple sclerosis drug.
  • peripheral blood mononuclear cells PBMCs
  • lymphocytes monocytes
  • macrophages macrophages
  • basophils dendritic cells or other cells derived from a mammal's blood.
  • a "reference standard” is a sample or value which serves as a point of comparison for another sample or value which differs from the reference standard with respect to one or more variables.
  • a “reference standard” is a value or range of values that characterizes a defined population in a defined state of health.
  • a reference standard can characterize a healthy human or a human afflicted with multiple sclerosis, and when the human is afflicted with multiple sclerosis the human can be naive or having received glatiramer acetate drug substance.
  • glatiramer acetate related drug substance is intended to include include polypeptides with a predetermined sequence as well as mixtures of polypeptides assembled from the four amino acids glutamic acid (E) , alanine (A) , lysine (K) , and tyrosine (Y) from any three of the amino acids Y, E, A and K, i.e. YAK, YEK, YEA or EAK; or from three of the amino acids Y, E, A and K and a fourth amino acid.
  • GERADS glatiramer acetate related drug substance
  • Glatiramer acetate related substances include glatiramoids .
  • a "glatiramer acetate related drug product" (GARDP) contains a glatiramer acetate related drug substance.
  • glatiramer acetate related drug substance or drug product is a glatiramer acetate related drug substance or a glatiramer acetate related drug product.
  • glatiramoid is a complex mixture of synthetic proteins and 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.
  • glatiramoids include glatiramer acetate drug substance (e.g. Copaxone ® ) as well as glatiramoids other than Copaxone ® , e.g. GA-Natco.
  • glatiramer acetate drug substance is glatiramer acetate produced by Teva Pharmaceutical Industries, Ltd. and is the active ingredient in a glatiramer acetate drug product.
  • a "glatiramer acetate drug product” (GADP) contains a glatiramer acetate drug substance produced by Teva Pharmaceutical Industries, Ltd.
  • Copaxone ® is a glatiramer acetate drug product.
  • glatiramer acetate drug substance or drug product is a glatiramer acetate drug substance or a glatiramer acetate drug product.
  • glatiramer acetate reference standard is or contains the drug substance found in a glatiramer acetate drug product .
  • suboptimal activity refers to a negative response or to a response which is less than the response to glatiramer acetate drug substance or glatiramer acetate drug product produced by Teva Pharmaceutical Industries, Ltd.
  • release of a drug product refers to making the product available to consumers .
  • about 100 mg therefore includes the range 90-110 mg and therefore also includes 90, 91, 92, 93, 94, 95 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109 and 110 mg. Accordingly, about 100 mg includes, in an embodiment, 100 mg.
  • hematological cell comprises neutrophils, erythrocytes, basophils, monocytes, eosinophils, platelets, lymphocytes, and splenocytes .
  • an "array of testing" for a glatiramer acetate related drug substance or drug product includes, but is not limited to, any analytical method test such as in vitro tests or molecular weight tests, biological assays such as the ex vivo tests and clinical efficacy tests which characterize the GARDS or GARDP, or clinical trials .
  • Examples of testing for a glatiramer acetate related drug substance or drug product are disclosed in U.S. Patent Application Publication Nos . US 2012-0309671 and US 2011-0230413, and in PCT International Application Publication Nos. WO 2000/018794, WO 2012/051106, WO 2013/055683, WO 2014/058976, the disclosures of which are hereby incorporated by reference in their entireties .
  • 0.2-5 mg is a disclosure of 0.2 mg, 0.21 mg, 0.22 mg, 0.23 mg etc. up to 0.3 mg, 0.31 mg, 0.32 mg, 0.33 mg etc. up to 0.4 mg, 0.5 mg, 0.6 mg etc. up to 5.0 mg.
  • characterization or “characterizing” is understood to include obtaining information which was produced in the same location or country, or a different location or country from where any remaining steps of the method are performed.
  • GA glatiramer acetate
  • mice were sacrificed and cells from their spleens (splenocytes) were isolated because such cells are representative of, and commonly utilized as a gold standard to study the immune system.
  • the aqueous activator samples, mannitol (the non-active excipient in Copaxone ® and all other marketed proposed generics) and medium were used as negative controls.
  • a total of 21 samples of Copaxone ® from 21 different batches were used, along with 10 samples from 2 lots of Escadra, 12 samples from 4 batches of Natco, 4 samples from 1 batch of Probioglat, 5 samples of Hangzhou from 2 batches, 12 samples of GA-RS from 1 batch, and 8 samples of medium.
  • RNA Isolation and Microarray Expression Profiling Extraction of total RNA from activated splenocytes was extracted using PerfectPure RNA Cultures CEKK kit 50 (5Prime GmbH, Hamburg, Germany) and following the manufacturer's instructions. RNA quality was assessed using the absorbance ratio at 260/280 ran and gel electrophoresis (Experion, Bio-Rad, Hercules, CA, USA) . Total RNA extracted from samples was hybridized to Illumina Mouse WG-6_V2 microarray chips containing more than 45,200 transcripts.
  • ComBat is an empirical Bayesian approach utilizing location and scale metrics across several genes to adjust for batch effects in datasets, even datasets containing small sample sizes. Treatment labels were added as covariates to the batch correction in order to preserve relevant treatment effects. Principal Component Analysis (a multivariate approach) showed that the main effect in the first principal component remained due to treatment effects after batch correction.
  • Mouse Splenocytes Gene Expression Analysis - Polimunol versus Copaxone ® Gene Expression Studies
  • GA-RS GA reference standard
  • phosphate-buffered saline 250 ⁇ ig GARS per mouse
  • GA-RS Teva
  • Polimunol is a proposed generic manufactured a company other than Teva.
  • mice were housed for 3 days after immunization; mice were then sacrificed and their spleens were aseptically removed and placed on ice in RPMI 1640. A single cell suspension was prepared. After red blood cells lysis, splenocytes from the same immunization group were pulled and resuspended to a final concentration of 10 x 106 cells/mL in defined cell culture media (DCCM1) (Biological Industries, Beit Haemek, Israel) (96.7% v/v) enriched with L-glutamine 2 mM (1% v/v), MEM Non-Essential Amino Acids 2 mM (1% v/v), sodium pyruvate 1 mM (1% v/v), antibiotic/antimycotic solution (0.2% v/v) and 2-mercaptoethanol 50mM (0.1% v/v).
  • DCCM1 defined cell culture media
  • Splenocytes were treated with activator samples diluted in medium (125 ⁇ per well of 80 pg/mL solutions, final concentration in the well: 40 g/mL) of: i) 3 different batches of GA drug product manufactured by Teva ii) one batch of proposed generic (Polimunol) .
  • Polimunol is a product marketed as generic GA and manufactured by a company other than Teva (i.e. Synthon) .
  • the activator samples, mannitol (the nonactive excipient in Copaxone ® ) , and medium were added to 96-well tissue culture plates (three wells per sample) .
  • Splenocytes (125 pL 10 x 10 s SPL cell/mL suspension) were added to the activator solutions. Each activator sample was loaded in two different plates. One for the cells from mice immunized with GA and one for cells from mice immunized with proposed generic. Plates were incubated for 24 h at 37 °C. Cells were collected from the wells and were centrifuged at 300g for 5 minutes. Supernatants were aspirated and cell pellets were resuspended in RLT buffer (from RNeasy mini kit of Qiagen, Cat # 74106) for cell lysis. The cell lysates were centrifuged and supernatants were collected and frozen at -70°C. Samples were sent for further processing .
  • mice were immunized with either Copaxone ® or Polimunol, and subsequently splenocytes were isolated and treated ex vivo with Copaxone 1® or Polimunol.
  • RNA was extracted and expression profiled across the entire genome using the Affymetrix Mouse Genome 430 2 chip. Three lots of Copaxone ® , and one lot of Polimunol were comparatively tested in six replicates each.
  • Outlier samples were identified using the R package ArrayQCMetrics and excluded from further data processing steps. A sample was considered an outlier if it ' failed more than half of the included tests either before or after RMA normalization. Data were RMA normalized using the Affy R package.
  • probesets were identified across conditions using linear models for microarray data (LIMMA; Smyth, G. K. (2004)), a standard R Bioconductor package. Linear models and empirical Bayes methods for assessing differential expression in microarray experiments. Statistical Applications in Genetics and Molecular Biology 3, No. 1, Article 3) .
  • Comparisons were corrected to compare each treatment relative to mannitol control (e.g., [GA vs mannitol] was compared via LIMMA to [Polimunol vs mannitol]).
  • Probesets were filtered by MAS5 calls of presence on the chip (to be considered present, a probeset was required to have on average a call of present or marginal across samples) . Probesets were mapped to genes using the annotation available for the Mouse 430 2 chip from Affymetrix. Unless otherwise noted, FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene. Pathway enrichment analysis
  • Upregulated and downregulated probesets were analyzed separately for pathway enrichment, using DAVID (Huang et al, Nucleic Acids Res 2009) . Pathway enrichment results were visualized using volcano plots, plotting either -log adjusted p values or untransformed adjusted p values versus enrichment scores for the pathways.
  • upregulated or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and fold changes (FC) with absolute value greater than 1.2 were used for pathway enrichment.
  • upregulated or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and FC with absolute value greater than 2 were used for pathway enrichment.
  • DAVID runs were conducted in December 2014 and January 2015. Human Monocyte Cell Line: Gene Expression Analysis
  • THP-1 human monocytes (TIB-202) were purchased from ATCC®. Cells were maintained in recommended RPMI-1640 media containing FCS, L-Glutamine, Sodium Pyruvate, D-Glucose, HEPES, and 2-mercaptoethanol at 37 °C and 5% C02. Prior to treatment cells were passed and plated in a 6-well plate at a concentration of 1.0 x 10 6 cells/mL. Cells were allowed to recover for four hours after passage (prior to treatment) .
  • RNA quality was assessed by determining the absorbance ratio 260/280nm as well as electrophoresis bioanalyzer with 260/280 ratio of 1.9-2.1 and RIN of above 9 were deemed acceptable.
  • Gene expression was measured using Affymetrix® U133 plus 2.0 format. Sample processing was executed according to established manufacturer protocols. The scheme in (Fig. 2) illustrates the general experimental outline. W
  • RNA from THP-1 cells was extracted and expression profiled across the entire genome using the Affymetrix Human Genome U133 Plus 2.0 chip, interrogating a total of over 47,000 transcripts.
  • Four batches of Copaxone ® and one batch of Probioglat were comparatively tested in six biological replicates each. Key identified genes were independently evaluated for level of gene expression by quantitative Real-Time PCR of samples collected in the same experiments.
  • Human Monocyte Cell Line Gene Expression Analysis - Polimunol versus Copaxone ® Gene Expression Studies
  • Outlier samples were identified using the R package ArrayQCMetrics and excluded from further data processing .steps. A sample was considered an outlier if it failed more than half of the included tests either before or after R A normalization. Data were RMA normalized using the Affy R package.
  • ComBat is an empirical Bayesian approach utilizing location and scale metrics across several genes to adjust for batch effects in datasets, even datasets containing small sample sizes. Date of experiment was utilized as batch. Treatment labels were added as covariates to the batch correction in order to preserve relevant treatment effects. Principal Component Analysis (a multivariate approach) showed that the main effect in the first principal component remained due to treatment effects after batch correction.
  • probesets were filtered by MASS calls of presence on the chip for the relevant samples in the comparison (e.g., to be considered present, a probeset was required to have on average a call of present or marginal across samples) .
  • An additional QC step was performed to remove probesets determined to be highly variable between multiple THP-1 datasets, as follows: a probeset was deemed highly variable if across three THP-1 studies to date, that probeset was observed to be upregulated, downregulated, and not modulated by Copaxone ® across the three studies. This criterion resulted in filtering out 216 probesets. Probesets were mapped to genes using the annotation available for the U133 Plus 2.0 chip from Affymetrix. FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene.
  • Upregulated and downregulated probesets were analyzed separately for pathway enrichment, using DAVID (Huang et al, Nucleic Acids Res 2009) . Pathway enrichment results were visualized using volcano plots, plotting -log p values versus enrichment scores. For comparisons between Copaxone ® and Polimunol, upregulated or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and fold changes (FC) with absolute value greater than 1.1 were used for pathway enrichment. For comparisons between Copaxone ® and mannitol, upregulated or downregulated probesets with FDR-adjusted p values ⁇ 0.05 and FC with absolute value greater than 1.3 were used for pathway enrichment. DAVID runs were conducted December 5 and 10, 2014.
  • THP-1 cells were activated with GA or Probioglat as described above.
  • the supernatant 1.0 mL of cell culture media
  • was collected at the 24 hour timepoint to account for the time duration required for translation relative to the 6 hour mRNA data reported herein) .
  • Luminex assay was utilized to measure the concentrations of a panel of 45 chemokines and cytokines (in pg/ml) using Bio-Plex Human Chemokine (Bio Rad kit) and Luminex Performance Assay (R&D kit) .
  • two other genes that were found to differ significantly between GA and Probioglat using the genome-wide microarray mRNA data were also present in the Luminex panel (CCL2, IL10) .
  • genes associated with monocytes in particular have previously been shown to be differentially expressed following treatment with a purported generic marketed by Natco in India, as compared with Copaxone (Huang et al . , Nucleic Acids Res. 2009) .
  • mRNA expression levels were compared between GA and control (mannitol) tested with 6 sample replicates for each of 4 batches of GA and for mannitol, using LIMMA (Sim et al., Nat. Biotechnol . 2011) (Methods).
  • Many genes were modulated significantly (FDR-adjusted p-value ⁇ 0.05 ) at each timepoint by treatment with branded GA (Table 1; Table 2 lists top modulated probesets) .
  • GA impacts expression most pronouncedly at 6h (Table 2) .
  • the use of this early timepoint may also be biologically relevant given that GA is thought to be rapidly degraded at the injection site, eventually without measurable blood levels (Vieira et al., J. Immunol. Baltim. Md 1950, 2003; Thamilarasan et al., J. Neuroinflammation 2013; Comi et al, Neurol 2002, Rizvi et al, Int J Nanomed 2006) .
  • the differentially expressed genes included several anti-inflammatory genes.
  • IL10 the gene encoding the anti-inflammatory cytokine IL-10
  • IL1RN encoding IL-lra, a protein that inhibits the activities of the pro-inflammatory cytokines IL-la and IL-lb
  • Fig. 4b- d shows the 6h timepoint for all present probesets (FDR adjusted p values 6.7e-16, 1.7e-10, and 1.2e-9, and FC 1.43, 1.35, and 1.26, respectively) .
  • top significantly up- and down- regulated genes were examined for pathway enrichment using DAVID as described in Methods ( Figure 5; Table 5) .
  • the top genes significantly upregulated by Copaxone ® in the human THP-1 cell line at 6 hours of treatment were enriched significantly (by Benjamini corrected p value ⁇ 0.05) for 114 pathways (Table 5), including many immune-related pathways ( Figure 5) .
  • the regulation of immune system process (GO : 0002682 ) and cytokine-cytokine receptor interaction (hsa04060) pathways were both significantly enriched among the top upregulated genes.
  • the top upregulated genes identified as members of the cytokine-cytokine receptor interaction pathway (hsa04060) are shown in Figure 6.
  • 9 pathways were significantly enriched among genes downregulated by GA (Table 5) .
  • PCDHGC3 /// PCDHGB4 /// PCDHGA8
  • PCDHGA11 /// PCDHGA10 ///
  • PCDHGA3 /// PCDHGA2 /// PCDHGAl
  • PCDHGC3 /// PCDHGB4 /// PCDHGA8
  • PCDHGC4 /// PCDHGB7 /// PCDHGB6
  • PCDHGA11 /// PCDHGA10 ///
  • PCDHGA3 /// PCDHGA2 /// PCDHGAl
  • GA is thought to induce an anti-inflammatory effect, mediated by secretion of IL-4, IL-10, and other anti inflammatory cytokines both in terms of T cells (Thl to Th2 shift) but also in terms of monocytes, resulting in a shift from monocyte production of IL-12 to anti-inflammatory IL-10.
  • monocytes from mice treated with GA secreted more IL-10 than monocytes from untreated mice (Weber, Nat Med 2007)
  • monocytes isolated from MS patients treated with GA were shown to produce more IL-10 relative to untreated patients (Kim, J Immunol 2004) .
  • dendritic cells exposed to GA during maturation increased their production of IL-10 (Mahad et al . Brain J. Neurol. 2006) .
  • IL1RN Another anti-inflammatory gene, IL1RN (encodinglL-lra, a protein that inhibits the activities of IL-la and IL-lb) showed increased expression at all three timepoints .
  • differential gene expression analysis was performed to compare directly between profiles induced by branded GA and by the purported generic glatiramoid, Probioglat.
  • the standard R LIMMA bioconductor package was utilized to measure differentially expressed probesets across the entire microarray.
  • comparisons were corrected to compare each treatment relative to mannitol control (i.e., [GA vs mannitol] was compared via LIMMA to [Probioglat vs mannitol] ) .
  • Probesets were filtered by calls of presence on the chip for the relevant samples in the comparison (to be considered present at a given timepoint, a probeset was required to have on average a call of present or marginal across the relevant samples at that timepoint) . Probesets were mapped to genes using the annotation available for the U133 Plus 2.0 chip from Affymetrix. FDR adjusted p values reported for genes represent the lowest FDR adjusted p value for present probesets for that gene.
  • A.at -1.137231 10.6086468 -6.1814333 3.02E-07 a0014O732 5.38434731.
  • P2RX4 20 oas_at 1.11462 10- 5804136 -5.05893O2 1-.06E-O5 0LO1O392&8 3.25362692
  • KYMU 2106 «3_5_at -1-1273404 10-5827862 -4.4730592 6.61E-05 0.03124079 1.64136616
  • OS8PU.1 218304_s_at -1.1200967 3.30361724 -4.268338 0.00012355 0.04461757 1.03859065

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EP15814349.5A 2014-07-01 2015-07-01 Caractérisation biologique d'un produit médicamenteux à l'acétate de glatiramère, à l'aide de cellules humaines et de mammifères Withdrawn EP3164710A4 (fr)

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WO2018053109A1 (fr) * 2016-09-14 2018-03-22 Teva Pharmaceutical Industries Ltd. Caractérisation d'expression génique d'un produit médicamenteux associé à l'acétate de glatiramère dans des cellules humaines et de mammifères
AU2018350693B2 (en) 2017-10-16 2021-03-04 F. Hoffmann-La Roche Ag Nucleic acid molecule for reduction of PAPD5 and PAPD7 mRNA for treating hepatitis B infection
CN116637123B (zh) * 2023-06-07 2024-02-13 上海市东方医院（同济大学附属东方医院） 敲降或下调C15orf39基因表达的试剂在制备治疗胃癌的药物中的应用

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TW201610169A (zh) 2016-03-16
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