bFGF GMP, human, recombinantTechnical Information

bFGF GMP, human, recombinant (146 aa)

Product Information
Molecular Mass
The 146 amino acid residues recombinant protein has a molecular mass of approximately 16,000Source
Escherichia coli

Concentration
1.0 mg/ml (Absorbance at 280 nm)

Buffer
20 mM phosphate, 2 M NaCl, pH7.5

Storage
Store at -70℃. Avoid repeated freeze-thaw cycles.

Filtrated by 0.2 μm filter
Animal component Free

Important NoticeThis product is manufactured in the facility to be operated in accordance with GMP for
Investigational Products (July 9, 2008, Pharmaceutical and Food Safety Bureau (PFSB) Notification
No. 0709002).
This product does not receive approval or clearance of any regulatory authority.
This product is for research use and ex vivo cell culture processing only, is not intended for human invivo applications.

Specification
This product is derived from E. coli, produced using non-animal reagents, and manufactured
in the facility to be operated in accordance with GMP for Investigational Products (July 9,2008, Pharmaceutical and Food Safety Bureau (PFSB) Notification No. 0709002). This productdoes not receive approval or clearance of any regulatory authority.
This product is for research use and ex vivo cell culture processing only, is not intended forhuman in vivo applications.
Test

Specification

ResultAnalytical Method

Appearance

Clear, Not visible particles

confirmed

Visual Inspection

Activity≧500,000 IU/mg≧800,000 IU/mg

Protein concentration

0.9～1.0 mg/mL

1.0 mg/mL

UV-Absorption (280nm)

Purity（SDS-PAGE）

Main band

confirmed

SDS-Polyacrylamide gel electrophoresis

Purity（HPLC）≧98%

99.80%

Gel filtration HPLC

Sterility Test

No microbial growth

confirmedEndotoxin≦10EU/mg＜0.5EU/mg

Balb 3T3 cell cell proliiferation assay, calibrated
against bFGF WHO International Standard (NIBSC)

Agar media culture: Thioglycollate medium andSoybean-Casein digest Medium
Kinetic Chromogenic LAL method : Kinetic-QCL
Reagent Kit

Residual DNA(E.coli) assay＜25 pg/mL

Q-PCR, Primer for 23s rRNA of Ecoli

Host Cell Protein(E.coli) assay＜1.0ng/mL

ELISA, HCP Ecoli ELISA Kit

SDS-PAGE, Sterility Test, Endotoxin, Residual DNA and Host Cell Protein analyses were performed under GLP.

Test Items List
Test

Analytical Method

Appearance

Protein concentration

Visual Inspection
Balb 3T3 cell cell proliiferation assay, calibrated against bFGF WHO
International Standard (NIBSC)UV-Absorption (280nm)

Purity（SDS-PAGE）

SDS-Polyacrylamide gel electrophoresis

Purity（HPLC）

Endotoxin

Gel filtration HPLC
Agar media culture: Thioglycollate medium and Soybean-Casein
digest Medium
Kinetic Chromogenic LAL method : Kinetic-QCL Reagent Kit

Residual DNA(E.coli) assay

Q-PCR, Primer for 23s rRNA of Ecoli

GLP

Host Cell Protein(E.coli) assay

ELISA, HCP Ecoli ELISA Kit

GLP

Activity

Sterility Test

condition
non-GLP
non-GLP
GLP
GLP
non-GLP
GLP
GLP

Protein Characterization
Peptid Mapping
Molecular weight
Cell Culture Test of iPS and ES cells
Undifferentiation
Karyotype analysis

Lys or Lys/trypsin digested sample were analized reverse phase
LC/MS and sequence assignment
sample were analized reverse phase LC/MS

GLP
GLP

Cells were cultured with bFGF GMP 5 passages
Nanog, oct3/4, rBC2LCN, SSEA4

G-bandEctoderm marker：Tuji-1 Mesoderm marker：Desmin Endoderm
Differential potential in three germ lineages
marker : AFP

non-GLP
non-GLP
non-GLP

Activity test
The specific activity of bFGF GMP human recombinant is approximately 3,000 kIU/mg,
which is calibrated against WHO International Standard (NIBSC code: 90/712).
bFGF GMP

bFGF IS

ED50=0.28

ED50=0.08

bFGF conc. (ng/mL)

bFGF conc. (ng/mL)

bFGF GMP human recombinant and bFGF WHO International Standard were tested by cell proliferation assay
using Balb-3T3 cell line. ED50 of bFGF GMP was 0.08 ng/ml which was over 3 times higher than bFGF IS(International Standard).

Purity test (SDS-PAGE)
12345625025018015015013010010010075757563503725483525205037252017151110LANE
1234561510Sample namePrecision Plus Protein Dual Colour Standards (Bio-Rad code:1610374)
Recombinant human FGF2 protein (Abcam, Cat. No. AB155734）
bFGF GMP human recombinant 3μg
HR Pre-Stained Protein Marker (10-170kDa) (BIOTOOLS, Cat. No. TM-PM10170)
bFGF GMP human recombinant 15μg
Precision Plus Protein Dual Colour Standards (Bio-Rad code:1610374)

4–15% Mini-PROTEAN® TGX™ Precast Protein Gels
CBB stain

ResultIn Molecular Mass analysis, themajor band of test article wasappeared at approximately 16 kDa.
(Precision Plus Protein Dual ColourStandards and HR Pre-StainedProtein Marker were used as
Molecular mass standards)

Purity test (HPLC: Gel filtration chromatography)
AU

Measurement method
Equipment ：HPLC system (Waters)
Column：TSKgel α-M (7.8×300 mm)
Mobile phase：10 mM-Tris 1M-NaCl HCl (pH7.4)
Detection wavelength：280 nm
Flow rate：0.6 ml/min
Apply volume：10 μl

17.640Peak No.

10.00

15.00

20.00

minRetention
time (min)Area%Area1

14.9041088118

99.796217.640

2219

0.204

Additional analytical Data
・Protein Characterization Analysis(GLP)・Peptide mapping by LC-MS analysis
・Molecular weight analysis by LC-MS・iPS・ES Cell culture testProtein Characterization
１．Peptide mapping
Analytical conditions＜Instrument＞
HPLC MS detector
MS detector：maXis impact
HPLC System：LC-30A System＜LC conditions＞
Column：Cadenza CD-C18（2 mm I.D.×150 mm, Imtakt
corp.）
Column Temperature: 50 °C
Flow Rate：0.2 mL/min
Detection wave length：214 nm
Mobile Phase A：water／formic acid（1000：1）
Mobile Phase B：Acetonitrile／water／formic acid
（800：200：1）
Injection Volume：5 μL

Gradient Conditions
time(min)059090.1
9595.1115

mobile phase A(vol%)
1001005500100100

mobile phase B(vol%)
0045100
10000＜MS condition＞
Ionization：ESI（Electrospray ionization）
Measurement ion：positive ion
Capillary Voltage：4500 V
Nebulizer：2.0 bar
Dry Gas：8.0 L/min
Dry Temp：220 °C
Detector Voltage：1823.2 V
Measurement range：m/z 50～2200＜MS/MS condition＞
Measurement range ：m/z 50～2200
MS/MS：AutoCollision Gas：NitrogenPeptide Mapping

Assignment result of LC-MS

LT digestion sample

LT digestion sample

cam-C : Carbamide methylated Cysteine
1)
The start time of MS integration
2)
Estimated that N(Asn) change to D(Asp) during enzyme digestion3)
Search by extract ion chromatogram peakAmino acid sequences and Assignment result：Assignment amino acid sequences and peak No. of MS fragment
1) Assignment as the non-specific digestion fragments
2) Estimated that N(Asn) change to D(Asp) during enzyme digestion

After Carbamide methylation, Samples digest LysC or LysC/Trypsin.
Main peak of peptide map of LT digestion was analyzed and assigned to AAsequence. Non assignment AA sequence for LT digestion map was assigned byextraction ion-chromatography analysis.
As the result, 2-147 AA sequence except for N-terminal AA(methionine) wasassigned.

２．Molecular Mass Measurement
Analytical conditions＜Instrument＞
HPLC MS detector
MS detector：maXis impact
HPLC System：LC-30A System＜LC conditions＞
Column：PLRP-S 300A 3um（1 mm I.D.×150mm，Agilent Technologies））
Column Temperature：75 °C
Flow Rate：0.2 mL/min
Detection Wave Length：280 nm
Mobile Phase A：water／formic acid（1000：1）
Mobile Phase B：Acetonitrile／water／formic acid
（800：200：1）
Injection Volume：10 μL

Gradient Conditiontime(min)041415192035

mobile phase A(vol%)9797
20119797

mobile phase B(vol%)
3380
99
9933

＜MS conditions＞
ionization：ESI （Electrospray ionization）
Measurement Ion：positive ion
Capillary Voltage：4500 V
Nebulizer：1.6 bar
Dry Gas：6.0 L/min
Dry Temp：220°C
isCID Energy：120 eV
Detector Voltage：1823.2 V
Measurement Range：m/z 1000～3500

Molecular Weight Measurement
Molecular weight data.
average molecular weightanalysis datetheoriticalmoleculra
weightdifferences(ppm)amino acidseaquence

16407.5

16407.8

-18.3

2-147

16310.3

16310.7

-24.5

3-147

The result of molecular weightmeasurement was 16407.5 and 16310.3 as
the average molecular weight. The each ofdata was 50 ppm difference between
theoretical molecular weight.
The C-terminal amino acid was thought
serine as the sequence information.

The result of Deconvolution Mass analysis of reduced samples

Cell culture test for iPS・ES cells
OutlineiPS Cell（201B7） and ES Cell（WA01） were cultured with bFGF GMP human recombinant(Katayama Chemical) 5 passages, Test for undifferentiation, Karyotype anddifferentiation potential in the three germ lineages
Test Conditions
media：TeSR-E6 (STEMCELL Technologies) + 2 ng/mL TGF-beta1 (R&D Systems)+ 10 ng/mL(iPS Cell) or 100 ng/mL (ES cell) bFGF GMP human recombinant(bFGF GMP, Katayama Chemical)
BD Matrigel GFR matrix (BD bioscience)
Undifferentiated marker
Nanog, oct3/4, rBC2LCN, SSEA4
Induce to the three germ lineages
Ectoderm marker：Tuji-1 Mesoderm marker：Desmin Endoderm marker : AFP
Karyotype Analysis
G-band（ NIHON GENE RESEARCH LABORATORIES Inc.）
Data was AIST Dr. Yuzuru ItoRefference ： Ito Y. et al,PLoS One. 2015 Apr 7;10(4):e0118931. doi: 10.1371

Human iPS Cells（201B7）
Undifferentiation markers detection of 0 passage

nanog

Oct3/4rBC2LCN

SSEA4

rBC2LCN

SSEA4

Undifferentiation markers detection of 5 passages

nanog

Oct3/4The iPS cells (201B7) keep undifferentiated status culturedwith bFGF GMP (Katayama Chemical)

Human iPS Cells（201B7）
Tree germ lineage after 5 passages

Tuj-1

DesminAFP

Differential potential after 5 passage cultured with bFGF GMP (Katayama Chemical)
was normal. Ectoderm marker：Tuji-1 Mesoderm marker：Desmin Endoderm marker : AFP
Karyotype of 0 passage

Karyotype of after 5 passages

No abnormality was observed for cultured with bFGF GMP (Katayama Chemical)

Human ES cells（WA01）
Undifferentiation markers detection of 0 passage

nanog

Oct3/4rBC2LCN

SSEA4

rBC2LCN

SSEA4

Undifferentiation markers detection of 5 passages

nanog

Oct3/4The ES cells (WA01) keep undifferentiated status cultured
with bFGF GMP (Katayama Chemical)

Human ES cells（WA01）
Tree germ lineage after 5 passages

Tuj-1Desmin

AFP

Differential potential after 5 passage cultured with bFGF GMP (Katayama Chemical)
was normal . Ectoderm marker：Tuji-1 Mesoderm marker：Desmin Endoderm marker : AFP
Karyotype of 0 passage

Karyotype of after 5 passages

No abnormality was observed for cultured with bFGF GMP (Katayama Chemical)