@Pupnolbut

TABLE OF'CONIENTS

Certificate of Analysis for Recombinant Human TNF-alpha

Analytical TestsN-terminal SequenceHPLCMass SpectrometryFinal SDS-PAGE gelBiological Assay

C onstruction of. E. coli expressing Human TNF- alpha

FIow Diagram

E.coh, DNA testing

Sterility Test

Mycoplasma Test

Certificate of Analysis for all Reagents used in fermentationfoldlng/processing and purification

@Pspnollhur

Certificate of AnalysisPeproTech's Recombinant Human TNF-c

Lot# 0609C25

Synonyms: Tumor Necrosis Factor, TNFSF2, Cachectin, Differentiation-inducing factor (DIF),Necrosin, Cytotoxin

Description: TNF-u is a pleiotropic pro-inflammatory cytokine secreted by various cells includingadipocytes, activated monocytes, macrophages, B cells, T cells and fibroblasts. It belongs to the TNF

family of ligands and signals through two receptors, TNFRI and TNFR2. TNF-o is cytotoxic to awide variety of tumor cells and is an essential factor in mediating the immune response against

bacterial infections. TNF-c also plays a role in the induction of septic shock, auto immune diseases,

rheumatoid arthritis, inflammation, atrd diabetes. Recombinant human TNF-cr is a soluble 157 aminoacid protein (17.4 kDa) which corresponds to C-terminal extracellular domain of the frrll lengthtransmembrane protein.

Product is produced using non-animal origin reagents.

Molecule: Recombinant Human TNF-o 17.4 V,Da I 157 alrino acid residues

Catalog Number: G-300-01A

Lot Number: 0609C25 Expiration Date: July 2014

Vial Size: 100pg

Sequence: VRSSSRTPSD KPVAHWANP QAEGQLQWLN RRANALLANG VELRDNQLW PSEGLYLIYS

QVLFKGQGCP STHVLLTHTI SRIAVSYQTK VNLLSAIKSP CQRETPEGAE AKPWYEPIYL GGVFQLEKGDRLSAEINRPD YLDFAESGQV YFGIIAL

Mycoplasma: Samples are examined in accordance with sTM V-611.3 for agar cultivableMycoplasmas. The assay includes the inoculation of the test article into broth and onto agar withsubsequent incubation at37oC under aerobic conditions, and also inoculation onto agar withincubation at37oC under anaerobic conditions. Negative results after 28 days.

PeproTech lnc., Princeton Business Park, 5 Crescent Ave., Rocky Hill, NJ 08553

{609) 497-0253 / Fax 6A9) 497-A321Email: info@peprotech.comwww.peprotech.com

@Pnpnollhcn

E.coli DNA: Sample is examined utilizing 32P-labeled E.coli DNA probe generated kom E.coligenomic DNA. The assay included the test article, test article spike, negative and positive controlsand standards. The test sample was calculated to contain less than 133.4 pg DNA/mt.

Sterility: Samples were performed in accordance with current USP guidelines for sterility. The testsample was examined for sterility by direct inoculation into fluid thioglycollate and soybean-caseindigest broths. The broth cultures were incubated and observed for growth for 14 days. No growthwas observed.

Endotoxin Level: Tested using the Limulus Amebocyte Lysate (LAL) Kinetic-Qcl test kit fromBioWhittaker. Final product endotoxin level is 0.007 EUipg.

N-terminal Sequencing (5 amino acids): V R S S S

Mass Spectrometryz 17310.85 daltons

Purity: > 98yo by SDS-PAGE gel and HPLC analyses.

Formulation: The sterile filtered solution was lyophilized from 10 mM Sodium Phosphate, pH7.4 +0.5 mM DTT.

Reconstitution: Centrifuge the vial prior to opening. Reconstitute in water to a concentration of 0.1-1.0 mg/ml. Do not vortex. This solution can be stored at 2-8oC for up to I week. For extendedstorage, it is recommended to further dilute in a buffer containing a carrier protein (example 0.1%BSA) and store in working aliquots at -20oC to -80oC.

Storage/Stability: The lyophilized protein is stable at room temperature for up to I month. Workingaliquots stored with a carrier protein are stable for at least 12 months at -20oC to -80oC. Avoidrepeated fu eezel thaw cycles.

Biological Activity: The EDso as determined by the cytolysis of murine L929 cells in the presence ofActinomyocin D is < 0.05 ng/ml, corresponding to a specific activity of> 2x 107 units/mg.

Country of Origin: USA

April30.2010Date

PeproTech Inc., Princeton Business Park, 5 crescent Ave., Rocky Hiil, NJ 0g553(609) 497 -0253 / Fax (609) 497 -0321Email: info@peprotech.comwww.peprotech.com

Kareri A. Pirog,Quality Assurance

Analgtical ReportTechnical Support

Peprotech Analytical Service

Protein SequencingH-TNF-alphaLot# 0609C25

July 10,2009

Sample of H-TNF-alpha Lot# 0609C25 was sequenced up to 6 cycles on an Applied Biosystems Procise 494sequencer.

User Sequence:

II-TIrIF-alphaLot # 0609C25

To:

From:

Subject:

Date:

Request Number:Date Received:Date Completed:

3237-070909-2July 9,2009JuIy 10,2009

Position:

Primary Sequence:Secondary Sequence:

The raw data and chromatograms are available

Sequence Expected: V R

d-9'"

I

VRSSSMVRSS

6

sofor your review.

SS

MAKINC ERT+'.

V&YTIC-.4ESERYTCES.'iN rvnLUArroN AND TESTTNC oF cyroKrNES

'3 t -:rta !'Princeton Business park . 5CrescentAve.,P.O.Box2TS.RockyHill,Newlersey0S553.BuildingB-1 .Tel:609-497-0253 ext. 150

070909-hTNFalpha-#0609C25-C-Cycle Yield 7/10/09, 6:48 AM

Uncorrected Yield (pmols) vs. Residue Number

Anc,latico/ ReptortTo: Technical Support Request Number: 3237-070909-2

From: Peprotech Analytical Services Date Submitted: July 9,2009Subject: HPLC Date Completed: July 10,2009

H-TNF-alphaLot#}609C25

Date: July 10,2009

Sample H-TNF-alpha Lot#0609C25 was analyzed by Agilent 1100 Series HPLC under the following conditions:

Column: Protein C4-2I4TPL04- Vydac

Gradient: 30%-70% Solvent B in 40 min.

Solvent A: 0.1%TFA in Water

Solvent B: 0.1%TFA in Acetonitrile

Flowrate: 1.0 ml/min, Detection: UV at 230nrrInjection Yolume: 15.0 ul

Concentration: 0.5 uglul

The raw data and fulIreport are available for your review

mAU

'yWD'1 A, Wavelength=230 nm (070909-2\004-0801.0 - 070903-2\001-0201.0)

q'=r,rera.)

'-1jr- / ir --'ill'I

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ooP-

ELUT|oN48% F 3 ELUT|oN.49%:l -iil-i;

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- io ___ 1s _ 20 _,_ 25 __--30 .- 35 40 -- , mrri

-60

.80

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H-TNF-alpha Lot#0609C25 G0809D:\...\Fi-TNF-alpha Lot#0609C25 7-9-09_0001.datAcquired: 13:45:00, July 09, 2009

Voyager Spec #1=>SMI9[BP = 17302.7 , 13411

f

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H-TN F-alpha Lot#0609C25 G0809

l

l

ll

34762.66

I

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11

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i17866.23

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'rlll'\w__*_.'u+0

27403-8

Mass (rn z)

45007.0

Date: March 4,2010

Protein: H-Activin-A, H-FGF-basic, H-TNF-alpha and H_EGF

Lane 1:

LxteZ: H-Activin-A Lor#1009C479 82310 (-)Lane 3: H-FGF-basic Lo#1009C08 K2309 Tris oLane 4: H-TNF-alpha Lor#0609C25 81910 (-)Lane 5: H-EGF Lot#1209C05 81910 (-)Lane 6: MarkerLane 7 : H-Activin-A Lo#1009C47 gB23 l0 (+)Lane 8: H-FGF-basic Lor#1009C08 K2301fris 1+;l-ane 9, H-TNF-alpha Lot#0609C25 81910 (+)Lane 10: H-EGF Lot#1209C05 81910 (+)Lane 1l:Lane 12:

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Cytotoxicity Assay for h-TNF-cl using 929 cellsSBH Sciences, lnc. - J.W./L.H./J.W. - July 15, 2009

c

C:ytokine: Human TNF+; PeproTech; Ca# G-300-01A; Lot# 0609C25 G0809All run in parallel:Human TNF-u,; PeproTech; Cat# AF-300-01A; Lot# 0609AFC25 G0809

Human TNF-a; PeproTech; Cat# 300-01A; LotE 0805CY25-2 F1609

Human TNF-o; PeproTech; Cat# G-300-0'lA; Lot# 0609C25 G0809

Procedurc: (Reconstituted in water)1) Resuspend 929 cells in culture media containing 10% HS (Gibco lot# 0892684).

Transfer l00ulAarell in assay plate (6,000 cellslrvell, passage# 2). lncubate overnight 37oC.2) Serial dilute h-TNF-ct in assay media containing 2pglml Actinomycin D (Sigma Cat #A-9415)3) Add 1OOpli\rell serial diluted h-TNF-cr to cells in assay plate. Final volumeftvell is 2001.t1 containing

1pg/ml Aclinomycin D, 10% Horse Serum and h-TNF-a as indicated below.4) Incubate 46 hours.5) Add 30pl of Promega Substrate CellTiter 96 Aqueous One Solution Reagent to each well.

6) lncubate 37oC and read OD at 490 nm,

Data:

Average Min.OD ( 11 .11 - 100.0 nglml ): 0.476Max Net O.D ( 0.00 ng/ml): 1132

Calculated Net O.D. for ED5g: 0.565

Result: ED56 is 0.033 - 0.049 ng/ml h-TNF-cr

Conc. h-TNF-cr O.D. readinq at 490nm after 6.2 hours RelativeActivitvno/ml Row F Row G Row H Ave. O.D. Net O.D.

lUU.UU33.33't1.11

3.701.23o.4'.1

0.140.050.02

u.4c60.481

0.4840.5140.536o.6240.7701.1021.380

u.4ti90.466

0.4870.5000.5330.5980.7591.0081.302

u.4/o0.4760.4850.5050.5380.6040.7080.971

1.140

U.4OU

0.4740.4850.5060.5360.6090.7461.0271.274

-U.UU6

-0.00'l

0.0100.03't0.0600.1334.2700.551

0 798

o.290.290.300.310.330.380.460.640.79

0.00

0.000.00

1.560

1.573't.652

1.687

1.6381.467

1.598

1.689

1.608 1j32

Gytotoxicity Assay for h-TNF-a using 929 cellsPeproTech; Caff G-300-01A; Lot# 0609C25 G0809

Ecoo{ooo2

1.30

1.10

0.90

0.70

0.50

0.30

0_ 10

-0.100.0'10 0.100 1.000 10.000

h-TNFc (ng/ml) flog scalel

Cytotoxicity Assay for h-TNF-s using 929 cellsSBH Sciences, lnc. - J.W./L.H./J.W. : July 15,2009

B

Cytokine: Human TNF-a; PeproTech; CaS 300-01A; Lo# 0805CY25-2 F1609All run in parallel:Human TNF-o; PeproTech; Cat# AF-300-01A Lot# 0609AFC25 c0809Human TNF-o; PeproTech; Cat# 300-01A; Lot# 0805CY25-2 Fl609Human TNF-o; PeproTech; Cat# G-300-01A; Lot# 0609C25 G0809

Procedure: (Reconstituted in water)1) Resuspend 929 cells in culture media containing 10% HS (Gibco lot# 0892684),

Transfer lO0ulArvell in assay plate (6,000 cells/well, passage# 2). lncubate overnight 37oC.2) Serial dilute h-TNF-cr in assay media containing 2pglml Actinomycin D (Sigma Cat #A-9415)3) Add lO0pllwell serial diluted h-TNF-cr to cells in assay plate. Final volume/well is 200p1 containing

'lpg/ml Actinomycin D, 10% Horse Serum and h-TNF<r as indicated below.4) lncubate 46 hours.5) Add 30pl of Promega Substrate CellTiter 96 Aqueous One Solution Reagent to each well.6) lncubate 37oC and read OD at 490 nm.

Data:

Result:

Average Min.OD ( 3.7 - 100.0 ng/ml ): 0.474Max Net O.D ( 0.00 ng/ml): 1.134

Calculated Net O.D. for EDs: 0.567

ED5s is 0.030 - 0.045 ng/ml h-TNF-cr.

Conc. h-TNF-cr O.D. readino at 490nm after 6.2 hours RelativeActivitynq/ml Row D Row E Ave. o.Lr. Net o.D.

100.0033.33

11 11

3.701.230.41o.140.05o.o2

0.4690.4730.473o.4u0.5250.5620.7511.014't.275

0.4690.464

0.4760.4830.5190.5810.7330.9561.236

0.4690.469

0.4750.4840.522o.5720.7420.9851.256

-0.005-0.005

0.0010.0100.0480.0980.2680.511

0.782

o.290.29

0.300.300.320.360.460.61

0.780.000.000.00

1.560

1.5731.652

1.687

1.6381.467

1.598

1.689

1.608 't.134

Gytotoxicity Assay for h-TNF-a using 929 cellsPeproTech; CaH 300-01A; Lo# 0805CY25-2 F{609

E

oo{oooz.

0_001 0.010 0.100 1.000

h-TNF{ (ngrml} flog scalel

10.000 100.000

Cytotoxicity Assay for h-TNF-cr, using 929 cellsSBH Sciences, lnc. - J.W./L.H./J.W. - July 15,2009

A

Cytokine: Human TNF-g; PeproTech; Cat# AF-300-01A; Lot# 0609AFC25 G0809

All run in parallel;Human TNF-g; PeproTech; Cat# AF-300-01A; Lot# 0609AFC25 G0809

Human TNF-g; PeproTech; Cat# 300-01A; Lot# 0805GY25-2 F1609

Human TNF-a; PeproTech; Cat# G-300-01A; Lot# 0609C25 G0809

Procedure: (Reconstituted in water)1) Resuspend 929 cells in culture media containing 10% HS (Gibco lot# 0892684).

Transfer lO0ulArell in assay plate (6,000 cellsArvell, passage# 2). lncubate overnight 37oC.

2) Serial dilute h-TNF-cr in assay media containing 2pg/ml Actinomycin D (Sigma Cat #A-9415)3) Add 100p1/well serial diluted h-TNF-o to cells in assay plate. Final volume/well is 200p1 containing

1pg/ml Ac{inomycin D, 10% l'lorse Serum and h-TNF-cr as indicated below.4) lncubate 46 hours.5) Add 30pl of Promega Substrate CellTiter 96 Aqueous One Solution Reagent to each well.

6) lncubate 37oC and read OD at 490 nm.

Data:

Average Min.OD ( 11.11 - 100.0 ng/ml ), 0.473Max Net O.D ( 0.00 ng/ml): 1.135

Calculated Net O.D. for EDse: 0.568

Result: EDso is 0.034 - 0.05'l ng/ml h-TNF-o

Conc. h-TNF-a O.D. readinq at 490nm after 6.2 hours RelativeActivityno/ml RowA Row B Row C Ave. O.D. Net O.D.

100.0033.33

11.113.701.23o.41o.140.050.02

U.4OU

0.466

0.4750.4890.5250.5930.7350.9821.214

U.4OU

0.475o.4760.5010.5320.6200.7661.0841.302

0.4710.481

0.4820.5040.5430.6100.7771.0791.285

0.4660.474

0.4780.4980.5330.6080.7591.0481.267

-0.0060.001

0.0050.0250.0610.1 350.2870.5760.794

0.290.29

0.300.310.330.380.470.650.79

0.00

0.000.00

1.560

1.5731.652

1.687

1.6381.467

1.598

1.689

1.608 1-135 1.00

Cytotoxicity Assay for h-TNF-a using 929 cellsPeproTech; Ga# AF-300-0{A; Lot# 0609AFC25 G0809

E

o@*oooz

1.30

1. t0

0.90

0.70

0.50

0.30

0.10

-0.100.000 0.001

Cytotoxicity Assay for h-TNF-cr using 929 cellsSBH Sciences, lnc. -- J.W./L.H./J.W. - July 15,2009

Summary Graph

no/ml

Human TNF-a;PeproTech; Cat*

AF-3OGO1A;Lot# 0609AFC2€

G0809

Human TNF-a;PeproTech; Cat#

300-01A; Lot#0805cY25-2

F1609

Human TNF-a;PeproTech; Cat#G-300-01A; Lot#0609c25 G0809

100.0033.3311.113.701.230.410.14

0.050.02000

u.4t,tt0.474o.4780.4980.5330.6080.7591.0481.2671.608

u.40v0.4690.4750.4840"5220.5720.742

0.985't.2s61.608

0.4680.4740.4850.5060.5360.6090.7461.O271.2741_608

E

6o!too

1.70

-

T-.1.50 l

.*-

130 |1.10

1 --0.90 +--- --

Cytotoxicity Assay for h-TNF-a using 929 cellsSummary Graph

0.010 0.100 1.000

h-TNF-a (ngrml) flog scalel

--.- Human TNF-a; PeproTech; Cat# AFsi. Human TNF-a; PeproTech; Catll 300-01A; Lot# 0805CY25-2 F1 609

:*:lly!E! ttl-3f.9eroE9h-; catll G--300-01A; Lot# 060ec25 G080e

0.70 +- --- --0.50

0.300.000 0.001 100.000

Sequence Name: TNFaPV2KLength:4616

GAATTCCGCT

GTGATAAATT

GGAATAACAT

AAGGTCAGCT

GTACCTTCTG

GAACTACACC

GTGAAACTCC

GACCGTCTGT

ACTGTAATAA

TTAATCGCCT

TTGCGCAGCC

GTGCACTCTC

GACGGGCTTG

CCGTCATCAC

ATACCAGGCC

AGTTGGTGAT

GCAAAAGTTC

ATCATGAACA

GCTCGAGGCC

GGTGCGACAA

TGATGTTACA

CTCCTGATGA

GGTGAAAATA

CGATCGCGTA

GTAATGGCTG

GGTGATTTCT

AGACCGATAC

AATATGGTAT

AATTGGTTGT

TTGAAGGATC

CACCTACAAC

CAGCAACACC

CATCCGGCAG

AAGCTCGACC

CTCACCTACC

ATCTCTGGCG

ATGGTACGTA

GCAGTGGCTG

AAGGTCTGTA

ATCAGCCGTA

CGAGGGTGCA

CCGCAGAAAT

TAAGGATCCA

TGCAGCACAT

TGAATGGCGA

AGTACAATCT

TCTGCTCCCG

CGAAACGCGC

TGAATCGCCC

TTTGAACTTT

GATTTATTCA

ATAAAACTGT

GCGATTAAAT

TCTATCGATT

GATGAGATGG

TGCATGGTTA

TTGTTGATGC

TTTCGTCTCG

GCCTGTTGAA

CACTTGATAA

CAGGATCTTG

TGATAATCCT

AACACTGGCA

AGATCACGCA

AAAGCTCTCA

TTCTTCACGA

TTCAACAGAT

GTCTTGGCCG

AAACAATGCC

GTGTTGACAT

GCTCCTCTCG

AACCGTCGCG

CCTGATCTAT

TTGCTGTATC

GAAGCGAAAC

CAACCGTCCT

GCTTGGCACT

CCCCCTTTCG

ATGGCGCCTG

GCTCTGATGC

GCATCCGCTT

GAGACGAAAG

CATCATCCAG

TGCTTTGCCA

ACAAAGCCAC

CTGCTTACAT

TCCAACATGG

GTATGGGAAG

TCAGACTAAA

CTCACCACTG

GCTGGCAGTG

CTCAGGCGCA

CAAGTCTGGA

CCTTATTTTT

CCATCCTATG

GATATGAATA

GAGCATTACG

TCTTCCCGAC

TCAACCGTGG

GGCAGACCTC

CGGGAAGGGC

CGACACCGCC

CCCCTGCAAA

AAATACCACT

CACTCCGTCC

CTAACGCCCT

TCTCAAGTAC

TTACCAGACC

CATGGTATGA

GACTATCTAG

GGCCGTCGTT

CCAGCTGGCG

ATGCGGTATT

CGCATAGTTA

ACAGACAAGC

GGCCCAAGCT

CCAGAAAGTG

CGGAACGGTC

GTTGTGTCTC

AAACAGTAAT

ATGCTGATTT

CCCGATGCGC

CTGGCTGACG

CGATCCCCGG

TTCCTGCGCC

ATCACGAATG

AAGAAATGCA

GACGAGGGGA

GAACTGCCTC

AATTGCAGTT

CTGACTTGAC

AACGCAGACC

CTCCCTCACT

AGCGCTCAAA

TGGATTTGCT

GACATGATCC

AAATAAATTC

GGCGGTGATA

GATAAGCCGG

GCTGGCAAAC

TGTTCAAGGG

AAAGTTAACC

ACCGATCTAC

ATTTCGCTGA

TTACAACGTC

TAATAGCGAA

TTCTCCTTAC

AGCCAGCCCC

TGTGACCGTC

AGCGCTGAGG

AGGGAGCCAC

TGCGTTGTCG

AAAATCTCTG

ACAAGGGGTG

ATATGGGTAT

CAGAGTTGTT

GAATTTATGC

GAAAACAGCA

GGTTGCATTC

AATAACGGTT

TAAGCTTTTG

AATTAATAGG

GGTGAGTTTT

TCATTTGATG

GGGACGGCGG

GTTCCGTGGC

TTCTGGCTGG

GATGCAGGGG

GAGGATGAAG

A,ACTGATAAA

ATATAAAAAA

CTGAGCACAT

TTGCTCATGT

GGCGTTGAGC

TCAGGGCTGC

TGCTGAGCGC

CTGGGTGGCG

ATCTGGCCAG

GTGACTGGGA

GAGGCCCGCA

GCATCTGTGC

GACACCCGCC

TCCGGGAGCT

TCTGCCTCGT

GGTTGATGAG

GGAAGATGCG

ATGTTACATT

TTATGAGCCA

AAATGGGCTC

TCTGAAACAT

CTCTTCCGAC

TTCCAGGTAT

GATTCCTGTT

TGGTTGATGC

CCATTCTCAC

TTGTATTGAT

CTCCTTCATT

CTCGATGAGT

CTTTGTTGAA

AAAGCAAAAG

ATGATGGGGC

TAAAAGCTAA

GTGGAGGAAG

AGAGTTTGAT

CATACAGATA

CGATGTTAAC

AGTTGCTMC

TCCGTGATAA

CCGTCGACTC

TATCAAGTCT

TATTTCAACT

GTGTACTTCG

AAACCCTGGC

CCGATCGCCC

GGTATTTCAC

AACACCCGCT

GCATGTGTCA

GAAGAAGGTG

AGCTTTGTTG

TGATCTGATC

GCACAAGATA

TATTCAACGG

GCGATAATGT

GGCAAAGGTA

CATCAAGCAT

TAGAAGAATA

TGTAATTGTC

GAGTGATTTT

CGGATTCAGT

GTTGGACGAG

ACAGAAACGG

TTTTCTAATC

TAAATCGAAC

TTCAAAATCA

GATTCAGGCC

CCGCATCTTT

GTGATGTCAT

GCTCAGGGTG

EiiACCATCTGCG

t^h

TCTAGAAGGA?-4a

CCTCAGGCAG32C

CCAGCTCGTG

ATGTTCTGCT4at I

CCGTGCCAGC

GGAGAAAGGTb}i)

GTATTATCGC: t4

GTTACCCAAC

TTCCCAACAGf,4!,

ACCGCATATG!, i) i.'

GACGCGCCCT.! {)4,J

GAGGTTTTCA

TTGCTGA;iT

TAGGTGGACCi:s,

CTTCAACTCA

AAAATATATC

GAAACGTCTT

CGGGCAATCAi rt ,t_,

GCGTTGCCAA

TTTATCCGTA

TCCTGAl-T.i1 84*

CTTTTAACAG1 9?i

GATGACGAGC

CGTCACTCAT2t$c

TCGGAATCGC?76i

CTTTfiCAAA..\ a I t\

AGAATTGGTT

TTTTGCTGAG) !1\;l

CCAACTGGTCa -1;;

TGGTATGAGT1\ r,6a

ACCGACAAGG

TCTGGTGAAG

TAGCGGTTCG

Page 1 of 2 DS Gene 61121201212:33:37 PM

Sequence Name: TNFaPV2KLength:4616

GTTTATTGAC

AACGCCGGAG

TAAATACATT

GAGTATTCAA

TGGTGAAAGT

CTTGAGAGTT

ATAGACTGGA

ATCTGGAGCC

TCTACACGAC

TGGTAACTGT

GAAGATCCTT

AGATCAAAGG

GTGGTTTGTT

TGTTCTTCTA

TGTTACCAGT

CAGCGGTCGG

GCGTGAGCTT

GAGAGCGCAC

CGTCGATTTT

CTTTTGCTGG

GAGCTGATAC

AAACCGCCTC

GCGCAACGCA

GTGGAATTGT

GACGGGATCA

GATCTATCCG

CAAATATGTA

CATTTCCGTG

AAAAGATGCT

TTCGCCCCGA

TGGAGGCGGA

GGTGAGCGTG

GGGGAGTCAG

CAGACCAAGT

TTTGATAATC

ATCTTCTTGA

TGCCGGATCA

GTGTAGCCGT

GGCTGCTGCC

GCTGAACGGG

TGAGAAAGCG

GAGGGAGCTT

TGTGATGCTC

CCTTTTGCTC

CGCTCGCCGC

TCCCCGCGCG

ATTAATGTGA

GAGCGGATM

GTACCGACGG

GAGACGTCAG

TCCGCTCATG

TCGCCCTTAT

GAAGATCAGT

AGAACGTTGC

TAAAGTTGCA

GGTCTCGCGG

GCAACTATGG

TTACTCATAT

TCATGACCAA

GATCCTTTTT

AGAGCTACCA

AGTTAGGCCA

AGTGGCGATA

GGGTTCGTGC

CCACGCTTCC

CCAGGGGGAA

GTCAGGGGGG

ACATGTTCTT

AGCCGAACGA

TTGGCCGATT

GTTAGCTCAC

CAATTTCACA

TGATATGGGG

GTGGCACTTT

AGACAATAAC

TCCCTTTTTT

TGGGTGCACG

GCAAACTATT

GGACCACTTC

TATCATTGCA

ATGAACGAAA

ATACTTTAGA

AATCCCTTAA

TTCTGCGCGT

ACTCTTTTTC

CCACTTCAAG

AGTCGTGTCT

ACACAGCCCA

CGAAGGGAGA

ACGCCTGGTA

CGGAGCCTAT

TCCTGCGTTA

CCGAGCGCAG

CATTAATGCA

TCATTAGGCA

CAGGAAACAG

CAAATGGTGG

TCGGGGAAAT

CCTGATAAAT

GCGGCATTTT

AGTGGGTTAC

AACTGGCGAA

TGCGCTCGGC

GCACTGGGGC

TAGACAGATC

TTGATTTAAA

CGTGAGTTTT

AATCTGCTGC

CGAAGGTAAC

AACTCTGTAG

TACCGGGTTG

GCTTGGAGCG

AAGGCGGACA

TCTTTATAGT

GGAAAAACGC

TCCCCTGATT

CGAGTCAGTG

GCTGGCACGA

CCCCAGGCTT

CTATGACCAT

TCACCATCCT

GTGCGCGGAA

GCTTCAATAA

GCCTTCCTGT

ATCGAACTGG

CTACTTACTC

CCTTCCGGCT

CAGATGGTAA

GCTGAGATAG

ACTTCATTTT

CGTTCCACTG

TTGCAAACAA

TGGCTTCAGC

CACCGCCTAC

GACTCAAGAC

AACGACCTAC

GGTATCCGGT

CCTGTCGGGT

CAGCAACGCG

CTGTGGATAA

AGCGAGGAAG

CAGGTTTCCC

TACACTTTAT

GATTAC

GTCGGCTGTG

CCCCTATTTG

TATTGAAAAA

TTTTGCTCAC

ATCTCAACAG

TAGCTTCCCG

GGCTGGTTTA

GCCCTCCCGT

GTGCCTCACT

TAATTTAAAA

AGCGTCAGAC

AAAAACCACC

AGAGCGCAGA

ATACCTCGCT

GATAGTTACC

ACCGAACTGA

AAGCGGCAGG

TTCGCCACCT

GCCTTTTTAC

CCGTATTACC

CGGAAGAGCG

GACTGGAAAG

GCTTCCGGCT

:,:'5a,rt)

GCACAGGCTG

TTTATTTTTC: iiirl

GGAAGAGTAT

CCAGAAACGC31 2'

CGGTAAGATC) illi t

GCAACAATTA328t

TTGCTGATAAa': a t\

ATCGTAGTTA

GATTAAGCAT

GGATCTAGGT-tuuli

CCCGTAGAAA?i:^

GCTACCAGCGi; fdtl

TACCAAATAC

CTGCTAATCCSJ zi)

GGATAAGGCG

GATACCTACAlJCi'

GTCGGAACAG

CTGACTTGAG

GGTTCCTCCA'i J: L'

GCCTTTGAGT

CCCAATACGC.113'

CGGGCAGTGA,{}$0

CGTATGTTGT

Page 2 of 2 DS Gene 61121201212:33:37 PM

Plasmid Name:

Expression Plasmid TN Fa/PV?K

TNFa/PV2K

Kanamycin

Temperature Shift (30"C to 42oC)Induction Method:

Recommended Host Strain: PHSl

Description of Plasmid Construction:

ResidueNumbers Frrnr:f ion

1-663 Synthetic Expression FragmentPr- Promoter (1-150)RBS (151-170)Coding for Met- hTNFa (171-644)Termination Codon & Linker (645-663)

664-1072 Spacer

1073-1077 Linker

1078-2812 Antibiotic Resistance

2813-2822 Linker

2823-3145 Spacer

3146-4616 Replication Origin

Synthetic DNA

pUC18 Plasmid(Purchased Com mercially)

pUC 1 8 Residues:399 -1, 2686-2677

Synthetic DNA

pACYC177 Plasmid(Gift from New England Biolabs,lnc,)

p ACY C17 7 Residues: 1 588-3322

Synthetic DNA

pUC18 Plasmid(Purchased Commercially)

pUCl 8 Residues: 2622-2300

pUC18 Plasmid(Purchased Commercially)

pUCl 8 Residues: 1926-456

Sequence Name: TNFaPV2KLength:4616

(d83

I/

dEcoO 1 0'PsPOMhAPal:Nhel-Bmtl

urallt

5 rNraPV2K

Page I of I DS Gene 611212012 {2:32:52 PM

Sequence Name: TNFaEFLength: 663

TNFa Expression Fragment

GAATTCCGCTCTCACCTACCAAACAATGCCCCCCTGCAAAAAATAAATTCATATAAAAAACATACAGATAAI I ll I ll I I ll I I I I I ll llll I I I I I I I lt I I I I tl I I lt t I I I I I I tlt I I ttt tt tlt I ll I I I r lJt

10 20 30 40 50 60 70

Promoter

CCATCTGCGGTGATAAATTATCTCTGGCGGTGTTGACATAAATACCACTGGCGGTGATACTGAGCACAICGI lll I I I I lt I I I I I I ll ll I I I ll I tlll I I I I I I I tll I I I I tt I I lt I I I I I I I I lt ll I I ll I I lt I80 90 100 1 10 120 1 30 140

RBS

--------> MVRSSSRTPSDKPV

ATGTTAACTC-IAGAAGGAGGAATAACATATGGTACGTAGCTCCTCTCGCACTCCGTCCGATAAGCCGGTTGI I I I I I I lll I I I I I I lll ll I I I I ll ll ll I I I I tl lt I I tl I I I I ll ll I I I I I I lt I rt lt I I I lt I I1s0 160 170 180 190 zCA 21A

AH VVAN PAA EGA L QWL N R R AN A L L

CTCATGTAGTTGCTAACCCTCAGGCAGAAGGTCAGCTGCAGTGGCTGAACCGTCGCGCTAACGCCCTGCTGI I I I llll I I I I I lll ll I I I I I I I I llt tl I I I I I lt lt I tt tl I lt I I I I I llt ll I I I I lt I I lr rl I220 230 24A 250 260 27A 280

ANGVELRDNOLVVPS EGLYLIYSOGCAAACGGCGTTGAGCTCCGTGATAACCAGCTCGTGGTACCTTCTGAAGGTCTGTACCTGATCTATTCTCAI I ll I ll I I I I t I I I lll ll I I I I lll ll I I lt I I lt I tt I I I I I lt I I I I I I tt lt I lt I I I I lll I lt I290 300 310 320 330 340 350

VLFKGOGCPSTH VLLCoding Region

NYTI SRIAAGTACTGTTCAAGGGTCAGGGCTGCCCGTCGACTCATGTTCTGCTGAACTACACCATCAGCCGTATTGCTGI I ll ll I I I I I I I I ll ll ll I I ll lli ll I I tl I lt I tlr I I I I lt I I I I I I I I lll I tll tt I lt I I I I I360 370 380 390 400 410 420

VSYOTKVNLLSAI KSPCORETPEGTATCTTACCAGACCAAAGTTAACCTGCTGAGCGCTATCAAGTCTCCGTGCCAGCGTGAAACTCCCGAGGGT

Page I of2 DS Gene 6n212A1212:30:35 PM

Sequence Name: TNFaEFLength:663

I I I ll I ll I I I I I Il I t I I I I I I ll I I I I I I I I ll I I I I I I I I ll tt I I I I I I lt I I I I I I I tlr I I I I I I

430 440 450 460 470 480 4go

AEAK PWYE P I Y L GGVF Q L E KGDR L

GCAGAAGCGAAACCATGGTATGAACCGATcTACCTGGGTGGCGTATTTCAACTGGAGAAAGGTGACCGTcTI I ll I I ll ll I I ll I I I I I I I I ll I I I I I I I I lrr r lr r r I I lt I I I I I tt I lt I I I I I I tl lt lt I I I I I

500 510 52A 530 540 550 560

SAE I N RPDYL D FAE SGOVYFG I I

GTCCGCAGAAATCAACCGTCCTGACTATCTAGATTTCGCTGAATCTGGCCAGGTGTACTTCGGTATTAICGlltl I I ll ll I ll I I I ll ll I ll I I ll I I I I lr r r r r r r r l lt tl I I I I rt ll I I I I I lt tlt I I I I I tt I

570 580 590 600 610 620 630

TerminationAL/

II

C AC TGT AATAAT AAGGATC C AGC T

lr r I I I tl I I lt I lt I I I tt lt tt660650

Page 2 of 2 DS Gene 611212012 {2:30:35 PM

Host Strain PHS1An E.Coli K-12 Derivative

GenotJpe: F= mcrA mcrB IN(rmD rmE)l::Tn10{Hg'}

Phenotype: Increased resistance to HgCl2 (10ug/rnl) relative to W3110Resistant to bactreriophage lambda infection at 30oC.

Sensitive to Ampicillin (100 ug/ml), Kanamycin (25 u{r,r,l), Tetracycline(25 ug/ml), and Chloramphenicol (25 ug/rnl).

Construction of PHS1:

The host strain PHS1 was constructed by inserting the DNA containing the CI857/Cro regulatoryelements of bacteriophage-lambda into E.coli strain W3110, according to the methodologyoutlined in Herrero M., Journal of Bacteriology, Nov., 1990, p.6557-6567 andp.6568-6572.

Specific protocol for the preparation of Host Strain PHS 1 :

1) ADNAfragment(EcoRl-BamHl)consistingofnucleotides34499-39168ofbacteriophagelarnbda-Cl857 was excised from phage DNA and subcloned into the EcoRl-BamHl sites ofpUClSNot using the DH5cr host strain. The resulting plasmid was designatedlambdaRB/pUC18Not.

2) A DNA fragment (Notl) containing the Cl857lCro regulatory elements of bacteriophagelambda was excised from larnbdaRB/pUC18Not and subcloned into the Notl site of pUT/Hgusing Cl l8(lambda-pir) as the host strain. The resulting plasmid was designatedlambdaNot/pUT/Hg.

3) Plasmid lambdaNot/pUTlHg was transfected into E. Coli host strain W3110 using increasedresistance to 10 ug/ml HgCl2 as the initial screen for stable insertion of the transposon intothe host strain. The use of the mercury resistance screen was difficult due to some naturalresistance by the W3110 host strain to HgCl2 at the 10 ug/ml concentration. Ultimately, thestable insertion of the CI857 lCro regulatory elements was confirmed by resistance to lambdainfection at 30oC. and the ability of the resulting strain to regulate, by temperature shiftinduction, the expression of genes residing on plasmids and transcribed by the lambda P1

promoter. The resulting strain was designated PHS 1.

Source of Materials:

E.Coli strain DH5o and Lambda-Cl857 DNA were purchased commercially from LifeTechnologies, Inc..

Plasmids pUCl8Not, pUT/t{g, and E.Coli host C118(lambda-pir).were obtained from Dr. BurtEnsley, Envirogen, lnc., Princeton, NJ who obtained these materials from the lab authoring theaforementioned publication. E.Coli strain W3110 was obtained from Dr. Burt Ensley,Envirogen, Inc., Princeton, NJ.

#PrTnoilbcn

Flow ChartRecombinant Human TNF-alpha

Lot # 0609C25

Flow Diagram:

Fermentation

Cell Disruption and Clarifrcation

Ion Exchange Chromatogaphy

Ion Exchange Chromatography

Formulation and Lyophilization

-C{r-

Client:

Charles River Protocol Number:

Protocol Effective Date:

On-Test Date:

Results:

PeproTech lnc.P O Box 275,5 Crescent Ave.

Rocky Hill, NJ 08553

GP-V614.5

10 DEC 2009

08 MAR 2010

charles riverSUMMARY REPORT

Version 1

Determination of DNA Levels in Samples by Means of DNA Hybridization (E cotiDNA)

Exception Document(s):

None

This report summarizes testing performed at the Charles River Laboratories, lnc., Malvem, PA, facitity, which isoperated in compliance with the applicable requirements of the U.S. Food and Drug Administration's GoodManufacturing Practic,e regulations as found in Title 21 CFR Parts 210 and 211. There were no deviations from thetest method(s) that impacted the quality or integrity of the test result(s).

21r*6f2or aDate

'Vt\nA-azo, b

Sample ldentification SampleNumber

Assay Result

Unspiked Spike Recovery

Human TNF-a G-300-01A

Lot# 0609C25 81910518000

<133.4 pdmlDNA

104o/o

Revision History

Version 1 N/A

Page 1 of 1

35BTechnology Drive, Malvern, PA 19355 o 610.64A.4550 r FAX 6I O.BB9.9O28

\.c<rFcharles river

SUMMARY REPORTVersion 1

Sterility Testing of Final Containers and Biological products (Direct Method)

PeproTech lnc.

PO Box 275,5 Crescent Ave.Rocky Hill, NJ 08553

GP.V66O

06MAB2OOB

05MAR2010

This report summarizes testing performed at the Charles River Laboratories, lnc., Malvem, pA, facility, which isgPeraled in compliance wS. the applicable requirements of the U.S. Food and Drug Administration,s GoodManufacturing Practice regulations as found in Title 21 CFR Parts 210 and 211. There were no deviations from thetest method(s) that impacted the quality or integrity of the test result(s).

J?,WzonDate

tqru*-rlUh

Client:

Protocol Ntrmber:

Protocol Effective Date:

On-Test Date:

Results:

Sample ldenttficatlon SampleNumber Assay Resuh

Human TNF-a G-3OS01A Lot# 0609C2S 81910 sl8000 No Growth

Exceptio n Document(s):

None

Date

_ arufi{Lxt2

Revision History

Page 1 of 'l

35STechnology Drive, Malvern, PA 19355 r 610.640.4550 o FAX 610.889.9028

-C{-

Client:

Protocol Numbor:

Protocol Effective Date:

On-Test Date:

Rcults:

PeproTech lnc

PO Box 275, 5 Crescent Ave.Rocky Hill, NJ 08553

GP-V611.3

15 AUG 2007

02 MAR 2010

charles riverSUMMARY REPORT

Version {

"Polnts to Consided' Testing forthe Presence of Agar cultivable and NonCultivable Mycoplaemas

Exception Document(s):

None

This report summarizes testing performed at the Charles River Laboratories, lnc., Malvern, PA, facility, which isoperated in compliance with the applicable requirements of the U.S. Food and Drug Administration's GoodManufacturing Practice regulations as found in Title 21 CFR Parts 210 and211. There were no deviations from thetest method(s) that impacted the quality or integrity of the test result(s).

Sample ldentification SampleNumber

Asay ResultMycoplasma

Human TNF-a G-300-01A Lot# 0609C25 81910 518000 Not Detected

et ftpL?flaDate

0b tfrlzithDate

Date

Revlslon History

Version I N/A

Page 1 of 1

35STechnology Drive, Malvern, PA 19355 . 610.640.4550 . FAX 610.889.9028

9sPBecton Diekinson and CoryanyBD Diagaostic SystensPO Box 999Sparks MD 21152-0999 US

Certificate of Analysis

Page: 1 of 2

Product NareCatalog NumberBatch NumberExpiration Date

PAIL YEAST EXTRACT 1OKG

21"2730 Manufacture Date z 2008/07/1,282137 90201,3/06/30

01. Dehydrated Appearance: Light to medium beige, to medium tan,free-flowing, homog:eneous.

02. Solubility: 1 and 2% solutions, solub.l-e in distilled or deionizedwater.

03. Solut.ion Appearance 1%: Light-medium amber, c1ear, may have a veryslight precipitate.

04. Solut.ion Appearance 2eoi Medium amber, c1ear, may have a veryslight precipitate.

05. Coagulable Protein: Not present06. Cultural Response: A solution of 1% Yeast Extract plus 0.5? sodium

chloride was prepared and the pH was adjusted to '7 .2 t 0.2 usingdilute NaOH. The medium was di-spensed into tubes and sterilized.Tubes were inoculated with the test organisms and incubated at35 *. 2"C for 18-48 hours.

TEST ORGANISMS ATCC@ RECOVERYNeisseria meningitidis 13090 fair to goodStaphylococcus aureus 25923 goodStreptococcus pneumoniae 6305 good

07. There are no animal sourced ingredients or anj-mal- derived reagentsused in the manufacture of this product.

08. Residual Solvents (CPMP/ICH/283/95): Typical analysis for YeastExtract indicates that there is less then 3880 ppm of cyclohexane.No other solvents were detected during analysis.

The test methods for characteristics listed below are:Nitrogen (Kjeldahl)

Charact.eristic Unit Value Low].lmit Highl,imit.

pH, 25"C : 6.9Loss on Drying : % 3.0Ash : % 10.0Sodium Chloride : % 0.0

0.00.00.09.0

5.015. 0

5.012.0Nitrogen :

Bulk Lot Number :

t 11.0818429'7

The Batch Number on this certificate is synonymous with the Lot Numbershown on the product labe1"

Creation Date: 2008/08/L2 l6:41:36

/ S mf Dickinson and consrany

BD Diagmostic SystemsPO Box 999Sparks MD 21152-0999 US

Certificate of Analysis

Page:-2 of 2

Product NameCatalog NnrnlcerBatch NunberExpiration Date

PAIL YEAST EXTRACT 1OKG212730 Manufacture Date : 2008/07/1,282L37 9020L3/06/30

BD Diagnostic Systems (BDDS) is an ISO 13485:2003 and ISO 9001-:2000Registered facility. BDDS products are manufactured in facilitiesregistered with the United States Food and Drug Administration (FDA),and are regulated by the FDA's Quality System Regulations (QSRs) . Thisproduct met BDDS string:ent quality standards at time of batch/:.otrelease. Any test resul-ts reported on t.his certif icate were obtainedat. time of release.

,fohn GerlichVice President,QuaLity Management andRegrulatory CorylianceSigaature Date: 2O08/08/L2

Creati-on Date: 2008/08/12 1-6:.41 :36