Analysis and Puriﬁcation of Antibodies/Proteins/Peptides/Nucleic Acids

Bioseparation

http://www.ymc.co.jp

PB-0083E

Column Selection Guide
Column Selection Guide ・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・2
Features of Separation Modes ・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・3
Separation of Antibodies/Proteins/Peptides ・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 3, 4
Separation of Nucleic Acids・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・5

Columns/Packing Materials
Ion Exchange Chromatography Columns/Media
BioPro IEX Columns・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 6-8
BioPro IEX SmartSep Q/S and BioPro IEX Q/S・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 9-11Size Exclusion Chromatography Columns
YMC-SEC MAB・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 12,13YMC-Pack Diol・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 14,15
Hydrophobic Interaction Chromatography Column
BioPro HIC BF・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 16
Reversed-phase Chromatography Columns
YMC-Triart/YMC-Triart Bio・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 17-20

Preparative Systems/Columns
Glass Columns ECO PLUS・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 21
Biochromatography Systems/Columns・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 22

Contract Puriﬁcation
Contract Puriﬁcation ・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・
・ 23

1

Column Selection Guide
Column Selection Guide
Proteins
PeptidesIon
exchange

BioPro IEX Columns

For separation of charge variantsSizeexclusion

YMC-SEC MAB

For separation of aggregatesand fragments of MAbs
For separation of proteins,sugars, and oligonucleotides

Hydrophobic
interaction

BioPro HIC BF

For drug-to-antibody ratio (DAR)
analysis of antibody-drug
conjugates (ADCs)

Reversedphase

YMC-Triart C18/C8

For separation of biomoleculeswith molecular weight ofup to 30,000
For separation of biomoleculeswith molecular weight ofup to 150,000

YMC-Pack Diol

YMC-Triart Bio C4

HILIC

Nucleic
acids

YMC-Triart Diol-HILICFor separation of polar
compounds with poor retention
on reversed-phase columns

BioPro IEX Columns

For high resolution analysis

YMC-Pack Diol

For separation of biomoleculesby molecular weightIon
exchange

OligonucleotidesSizeexclusion

Oligonucleotides

Hydrophobic
interaction

Oligonucleotides

BioPro HIC BF

For separation of biomolecules
which is diﬃcult to be separated
by ion exchange chromatography

Reversedphase

Nucleic acid bases
Nucleosides
Nucleotides

YMC-Triart C18
Hydrosphere C18

Usable with 100% aqueous
mobile phase

Oligonucleotides

YMC-Triart C18/C8
YMC-Triart Bio C4

For high resolution analysis
Eﬀective for LC/MS

Nucleic acid bases
Nucleosides
Nucleotides

YMC-Triart Diol-HILIC
YMC-Pack Polyamine Ⅱ

For separation of polar compounds

HILIC

2

Features of Separation Modes
Ion
exchangeSizeexclusion

Hydrophobic
interaction

Reversedphase

BioPro IEX Columns

YMC-SEC MAB
YMC-Pack Diol

BioPro HIC BF

YMC-Triart etc.

Electric charge

Molecular weight

Hydrophobicity

Hydrophobicity

Principle ofseparation

Molecular
weight range

Up to several millions Up to about 1,000,000 Up to several millions Up to about 150,000

Resolution

+++

++

+++

+++

Speed

++〜+++

+

+++

+++

Loading

+++

++

+++

++

Samplestability

+++

+++

+++

+〜++

Typicalapplications

Charge variants
analysis

Separation of
aggregates
Separation offragments

Drug-to-antibody ratio
analysis of
antibody-drug
conjugates

Peptide mapping
LC/MS
Structural analysis

Separation of Antibodies/Proteins/Peptides
Charge variants analysis of monoclonal antibodies
Ion
exchange

BioPro IEX SF

5 µm, 100 X 4.6 mmI.D.

Salt gradient mode
1

2

4

3
1. Natalizumab (Humanized IgG4, pI=7.3)
2. Cetuximab (Chimeric IgG1, pI=7.9)
3. Adalimumab (Human IgG1, pI=8.4)
4. Denosumab (Human IgG2, pI=8.8)
Eluent: A) 10 mM MES-NaOH (pH 5.7)
B) 10 mM MES-NaOH (pH 5.7) containing 1 M NaCl
0-20%B (0-20 min)Flow rate : 0.6 mL/min

0

10

20 minpH gradient mode
1

2

3

4

Eluent: A) CX-1 pH Gradient Buﬀer A* (pH 5.6)
B) CX-1 pH Gradient Buﬀer B* (pH 10.2)0-100%B (0-20 min)Flow rate : 0.6 mL/min

0

10

20 min*Purchased from Thermo Fisher Scientiﬁc Inc.
J. Pharm. Biomed. Anal., 2015, 111, 169-176.

Monoclonal antibodies were separated by ion exchange chromatography in salt gradient mode and in pH gradientmode. Sharp peaks were obtained in both modes with BioPro IEX columns since they have extremely low
nonspeciﬁc adsorption. This shows that BioPro IEX columns are eﬀective for separation of charge variants andisoforms.

3Aggregates analysis of antibody-drug conjugate (ADC)
Size
exclusion

YMC-SEC MAB

3 µm, 300 X 4.6 mmI.D.

mAU
14

Eluent: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl/2-propanol (85/15)
Flow rate: 0.165 mL/min
Temperature : 25℃
Detection : UV at 280 nm
Injection: 4 μL
Sample: Cysteine-conjugated ADC mimic (2.5 mg/mL)

12
10
8
6
4

Size exclusion chromatography is useful for
separation of substances with diﬀerent
molecular weights such as MAbs, theirdimers and aggregates or ADCs, their dimers
and aggregates.

2
0
-2
-4

M180803EA

10

12

14

16

18

20

22

min

Drug-to-antibody ratio (DAR) analysis of ADC
Hydrophobic
interaction

BioPro HIC BF

4 µm, 100 X 4.6 mmI.D.

mV
30
20
10
0

L171130C

0

5

10

15

20 minEluent: A) 50 mM NaH2PO4-Na2HPO4 (pH 7.0) containing
1.5 M (NH4)2SO4/2-propanol (95/5)
B) 50 mM NaH2PO4-Na2HPO4 (pH 7.0)/2-propanol(80/20)0%B (0-1 min), 0-100%B (1-15 min), 100%B (15-20 min)Flow rate: 1.0 mL/min
Temperature : 25°
C
Detection: UV at 280 nm
Injection: 5 μL
Sample: Cysteine-conjugated ADC mimic (5 mg/mL)

Hydrophobic interaction chromatography
column BioPro HIC BF provides superior
separation for ADCs and is eﬀective for
DAR determination.

Peptide mapping
Reversedphase

YMC-Triart C18

1.9 µm, 200 X 2.0 mmI.D. (100 X 2.0 mmI.D. two coupled)
Eluent: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.08)
5-40%B (0-30 min)Flow rate: 0.4 mL/min
Temperature : 70°
C
Detection: UV at 220 nm
Injection: 20 μL
Sample: Triptic digest of Bovine Hemoglobin

P110707A

2

24 minThe outstanding eﬃciency obtained by acoupling of two 100 mm length of Triart
1.9 μm columns allows the precise
separation in peptide mapping.

Analysis of antimicrobial peptides
Reversedphase

YMC-Triart C18

1.9 µm, 50 X 2.0 mmI.D.

1. a-Defensin-1 : A CYCRIPACIAGERRYGTCIYQGRLWAFCC
2. a-Defensin-2 :CYCRIPACIAGERRYGTCIYQGRLWAFCC
3. a-Defensin-3 : D CYCRIPACIAGERRYGTCIYQGRLWAFCC

mAU
60

1

40

2

MW
MW
MW

3,442
3,371
3,486

3

20
0

F120501D

0

1

2

3

4

min

Eluent: A) water/formic acid (100/0.1)
B) acetonitrile/2-propanol/formic acid (50/50/0.08)
10-25%B (0-10 min)Flow rate: 0.4 mL/min
Temperature : 70°
C
Detection: UV at 220 nm

High temperature condition improves separationof peptides and proteins on reversed-phase
chromatography. Since Triart columns are highly
durable at high temperatures, they are eﬀective
for analysis of peptides and proteins under suchharsh conditions.

4Separation of Nucleic Acids
Analysis of oligonucleotides
Reversedphase

YMC-Triart C18

1.9 µm, 50 X 2.1 mmI.D.

mAU

1. 5ʼ-CAC UGA AUA CCA AU-3ʼ (14 mer)
2. 5ʼ-UCA CAC UGA AUA CCA AU-3ʼ (17 mer)
3. 5ʼ-UCA UCA CAC UGA AUA CCA AU-3ʼ (20 mer)
4. 5ʼ-GUC AUC ACA CUG AAU ACC AAU-3ʼ (21 mer)

4

15

Eluent

10

3

2

1

5

: A) 200 mM HFIP*-8 mM triethylamineB) methanol
10-20%B (0-10 min)Flow rate: 0.42 mL/min
Temperature : 65°
C
Detection: UV at 260 nm
Injection: 1.0 μL (2-4 nmol/mL)*1,1,1,3,3,3-hexaﬂuoro-2-propanol0

Triart C18 is suitable for separation ofhydrophilic compounds such as
oligonucleotides.

F180403J

02

Ion
exchange

4

6

BioPro IEX QF

8

10 min#5 µm, 100 X 4.6 mmI.D.

1. 5ʼ-U(M)C(M)A(M)U(M)C(M)A(M)C(M)A(M)C(M)U(M)G(M)A(M)A(M)U(M)A(M)C(M)C(M)A(M)A(M)U(M)-3ʼ (20 mer)
2. 5ʼ-G(M)U(M)C(M)A(M)U(M)C(M)A(M)C(M)A(M)C(M)U(M)G(M)A(M)A(M)U(M)A(M)C(M)C(M)A(M)A(M)U(M)-3ʼ (21 mer)
N(M)=2ʼOMe RNA

mAU

2

50

1

40

Eluent: A) 10 mM NaOH
B) 10 mM NaOH containing 1.0 M NaClO4
25-55%B (0-15 min), 100%B (15-20 min)Flow rate: 1.0 mL/min
Temperature : 25°
C
Detection: UV at 260 nm
Injection: 4 μL (5 nmol/mL)

30
20
10
0

P171116A

0

2

4

6

Plasmid pBR322
Size
exclusion

8

10

12

14 min

100 mm length column of BioPro IEX QF,
which has high separation ability, issuitable for separation of oligonucleotides
by one nucleotide diﬀerence.

Ⅲ restriction fragments

YMC-Pack Diol-300 + Diol-200

5 µm, 500 X 8.0 mmI.D. X 2

Plasmid pBR322 Hae Ⅲ digest (8-587 bp)

mAU

Plasmid pBR322(4,361 bp)

40
30
20
10
0

P080617A

0

10

20

30

40

50 minEluent: 0.1 M KH2PO4-K2HPO4 (pH 7.0) containing 0.2 M NaCl
Flow rate: 0.7 mL/min
Temperature : ambient (25°C)
Detection: UV at 260 nm
Injection: 10 μL

In size exclusion mode, separation is expected to be improved by using coupled columns with diﬀerent
pore sizes.

5Columns/Packing Materials
Ion Exchange Chromatography Columns for Separation of Proteins/Nucleic Acids

BioPro IEX Columns

Hydrophilic polymer beads with low nonspeciﬁc adsorption
Ideal for analysis and laboratory-scale puriﬁcation on porous type with highbinding capacity and high recovery of biomolecules
Ultra-fast analysis and high-resolution analysis on non-porous type
Suitable for characterization of biopharmaceuticals and quality control

Features

SEM images of polymer beads

Non-porous polymer beads

Porous polymer beads

Speciﬁcations
BioPro IEX QF
MatrixBioPro IEX SF

BioPro IEX QA

Hydrophilic non-porous polymer

Particle size (μm)

Hydrophilic porous polymer
53, 5Charged group

BioPro IEX SP+
3）
3−CH2N（CH−CH2CH2CH2SO3−+−CH2N（CH3）
3−CH2CH2CH2SO3−Counter ion

Cl−

Na+

Cl−

Na+

Ion exchange capacity*(meq/mL-resin)

0.09

0.24

0.09

0.09

Dynamic binding capacity*(mg/mL-resin)>12（BSA）>10（human-IgG）>110（BSA）>70（human-IgG）

Usable temperature

4 - 60℃

Usable pH range

2-12

Column material

PEEK*Reference value

High-resolution analysis of monoclonal antibody (MAb) 1〉
BioPro IEX SF

3 µm, 100 X 4.6 mmI.D.
DAD1 A, Sig=215,4 Ref=oﬀ (D:\DATA\抗体アプリ\170221-2 2017-02-21 16-02-42\U170221C3.D)

mAU
1200

Eluent: A) 20 mM NaH2PO4-Na2HPO4 (pH 6.8)
B) 20 mM NaH2PO4-Na2HPO4 (pH 6.8)
containing 0.2 M NaCl
0-50%B (0-30 min)Flow rate: 0.5 mL/min (180 cm/hr)
Temperature : 25°
C
Detection: UV at 215 nm
Injection: 10 μL
Sample: Humanized monoclonal IgG1 (2.5 mg/mL)

1000
800
600
400

BioPro IEX SF column with 3 µm,
100 mm length achieved high resolution
analysis of MAb.

200
0

U170221C3

5

10

15

20

25

min

6

High-resolution analysis of monoclonal antibody (MAb) 2〉
BioPro IEX SF

5 µm, 100 X 4.6 mmI.D.
mAU
3tR 60.0 min

mAU
12

2

8

1

4

0

30

V110221F

0
0

20

40

60

Competitorʼs WCX column

80

40

50

60

70

80 minmin

10 µm, 250 X 4.0 mmI.D.

mAU
3

mAU
12

2

8

1

4
0

V110223G

0tR 118.2 min

0
90

110

20

40

130

60

min

80

100

120

140 minEluent: A) 20 mM MES-NaOH (pH 5.6)
B) 20 mM MES-NaOH (pH 5.6)
containing 0.2 M NaCl
Initial gradient conc : 35%B (70 mM NaCl）
Gradient slope: 0.25%B/min (0.5 mM NaCl)
Flow rate: 180 cm/hr(0.5 mL/min for 100 X 4.6 mmI.D.,
0.378 mL/min for 250 X 4.0 mmI.D.)
Temperature: 30°
C
Detection: UV at 280 nm
Injection: 10 μL
Sample: Humanized monoclonal IgG1 (1 mg/mL)

The separation of MAb is compared on
BioPro IEX SF and competitorʼs column
under the same gradient conditions at
pH 5.6.
BioPro IEX SF column provides higher
resolution of MAb in a shorter analysis
time than the competitor's column.

Excellent batch‒to-batch reproducibility

ColumnEluent: BioPro IEX SF 5 μm, 100 X 4.6 mmI.D.
: A) 20 mM NaH2PO4-Na2HPO4 (pH 6.5)
B) 20 mM NaH2PO4-Na2HPO4 (pH 6.5)
containing 0.2 M NaCl
0-50%B (0.5-30 min)Flow rate: 0.5 mL/min (180 cm/hr)
Temperature : 25°
C
Detection: UV at 215 nm
Injection: 10 μL
Sample: Humanized monoclonal IgG1

Batch. E

Batch. D

BioPro IEX SF column exhibits excellent
batch-to-batch reproducibility for MAb
analysis, including the resolution of small
peaks for charge variants. All the medium
batches are inspected by various quality
control tests and must pass rigorous criteria
before release. BioPro IEX columns are thebest choice for the quality control of MAbs andother biopharmaceuticals.

Batch. C

Batch. B

Batch. A
0

5

10

15

20

25

30

min

High-resolution analysis of nucleic acids
BioPro IEX QF

5 µm, 100 X 4.6 mmI.D.

DNA fragments 1Kb DNA ladder (75-12,216 bp)
Eluent: A) 20 mM Tris-HCl (pH 8.1) containing 0.7 M NaCl
B) 20 mM Tris-HCl (pH 8.1) containing 1.0 M NaCl
0-100%B (0-30 min)Flow rate: 0.5 mL/min (180 cm/hr)
Temperature : 25°
C
Detection: UV at 260 nm
Injection: 20 μL (0.25 mg/mL)

mAU

50

0

N080212G

0

7

10

20 minThe separation of DNA fragments is shown.
BioPro IEX QF of 100 mm length column is agood choice for high-resolution analysis of
nucleic acids.

High binding capacity and recovery
Comparison of dynamic binding capacity (DBC) and recovery for BSA
Dynamic binding capacity（mg/mL-resin, 10% breakthrough）

Eluted amount（mg/mL-resin）

Recovery*（%）

BioPro IEX QA

126

120

95

Brand T (porous Q type)

73

58

79

Brand G (porous Q type)

100

35

35

*Recovery:（Eluted amount/Dynamic binding capacity）X 100

Breakthrough curves
Column: BioPro IEX QA, 50 X 4.6 mmI.D.
Brand T (porous Q type) 50 X 4.6 mmI.D.
Brand G (porous Q type) 50 X 5.0 mmI.D.
Linear velocity: 180 cm/hr
Equilibration buﬀer : 20 mM Tris-HCl (pH 8.6)
Elution buﬀer: 20 mM Tris-HCl (pH 8.6) containing 1.0 M NaCl
Detection: UV at 280 nm
Sample: 1 mg/mL Bovine serum albumin (BSA) in equilibration buﬀer

BioPro IEX QA

0.25

Brand G

Brand TAU
0.50

10% breakthrough

0
0

20

40
60
80
100
120
BSA amount loaded (mg/mL-resin)

140

160

BioPro IEX QA gives the superior DBC and recovery compared with conventional porous polymer anion exchange
columns. The surface structure of porous type BioPro IEX, which is designed for maximum interaction with proteins,provides high binding capacity, and the hydrophilic property of polymer beads signiﬁcantly reduces nonspeciﬁc
adsorption of proteins.

High loadability
Comparison of the eﬀect of sample load on BioPro IEX QA and commercial Q type column
BioPro IEX QA
5 μm, 50 X 4.6 mmI.D.

Brand G (porous Q type)10 μm, 50 X 5.0 mmI.D.

1

1. Ovalbumin
2. Trypsin inhibitor
2

mAU

mAU
1
Loadingamount200

2

200
500 μg500 μgN080206E

N080206B

100

250 μg

0
0

10

20

50 μg

0 N080206D

30 min0

1030 min20

Recovery of trypsin inhibitor

5×107

Eluent: A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 0.5 M NaCl
10-80%B (0-30 min)Flow rate: 0.5 mL/min (180 cm/hr for 4.6 mmI.D., 150 cm/hr for 5.0 mmI.D.)
Temperature : 25°
C
Detection: UV at 280 nm
Injection: 100 μL

BioPro IEX QA
Brand G
(porous Q type)

4×107
3×107
2×107

BioPro IEX QA shows excellent resolution and peak shapes evenwhen the loading amount increases. The porous type BioPro IEX
columns are suitable for laboratory-scale puriﬁcation of proteins.

1×107
0

100 μgN080208B

50 μg

N080206A

250 μgN080208C

100 μgN080207B

Peak area

100

N080207C

0

0.5

1.0
1.5
Loading (mg)

2.0

2.5

Ordering information
BioPro IEX QF/SF (non-porous type)

Packing
material
BioProIEX QF

Column size
Particle
size (μm) inner diameter X length (mm)35

BioProIEX SF35

4.6 X 30
4.6 X 50
4.6 X 100
4.6 X 30
4.6 X 50
4.6 X 100
4.6 X 30
4.6 X 50
4.6 X 100
4.6 X 30
4.6 X 50
4.6 X 100

BioPro IEX QA/SP (porous type)

Product number
QF00S03-0346WP
QF00S03-0546WPQF00S03-1046WPQF00S05-0346WP
QF00S05-0546WPQF00S05-1046WP
SF00S03-0346WP
SF00S03-0546WP
SF00S03-1046WP
SF00S05-0346WP
SF00S05-0546WP
SF00S05-1046WP

Packing
material

Particle
Column size
size (μm) inner diameter X length (mm)BioProIEX QA

5

BioProIEX SP

5

4.6 X 30
4.6 X 50
4.6 X 100
4.6 X 30
4.6 X 50
4.6 X 100

Product number
QAA0S05-0346WPQAA0S05-0546WPQAA0S05-1046WPSPA0S05-0346WPSPA0S05-0546WPSPA0S05-1046WP

8

High Throughput Ion Exchange Chromatography Media for Puriﬁcation of Biopharmaceuticals

BioPro IEX SmartSep Q/S
BioPro IEX Q/SFeatures

High productivity on puriﬁcation
Hydrophilic polymer beads with low nonspeciﬁc adsorption
High binding capacity and high resolution over a wide range of ﬂow rate
Suitable for puriﬁcation of antibodies, proteins and nucleic acids

Speciﬁcations
BioPro IEX SmartSep Q

BioPro IEX SmartSep S

MatrixBioPro IEX S

BioPro IEX Q

Hydrophilic porous polymer

Particle size (μm)
Charged group10, 20, 30

Ion exchange capacity(meq/mL-resin)
Dynamic binding capacity(mg/mL-resin)

75−R-SO3−+−R-N（CH3）3>0.08>100（BSA）−R-SO3−+3）
3−R-N（CH>0.10>160（BSA）>100（lysozyme）

Usable pH range>160（lysozyme）

2-12

High sample loadability
High dynamic binding capacity (DBC) for various samples
BioPro IEX SmartSep S30

120

Brand T
（porous S type 30 μm）
Brand G
（porous S type 30 μm）

DBC (mg/mL-resin)

100

Conditions of DBC measurement*
Column: 50 X 5.0 mmI.D.
Flow rate: 400 cm/hr (1.32 mL/min)
Temperature : 25℃

80
60*Please inquire us for details.

40
20
0

Insulin(MW : 5,800)

Lysozyme(MW : 14,300)

Human Polyclonal IgG(MW : 150,000)

Particlesize（μm）

InsulinLysozyme

Human Polyclonal IgG

BioPro IEX SmartSep S30

30

73

111

93

Brand T
（porous S type）

30

67

72

42

Brand G
（porous S type）

30

64

85

41

DBC
（mg/mL-resin, 10% breakthrough）

BioPro IEX media have higher DBC compared to conventional ion exchange media. Especially for IgG, BioPro
IEX media have more than twice as high DBC as competitors' media. This feature of BioPro IEX media makepuriﬁcation productivity of IgG per unit time double or more.

High dynamic binding capacity (DBC) over a wide range of ﬂow rate
120
110

BioPro IEX SmartSep S30

DBC (mg/mL-resin)

100

Brand T (porous S type 30 µm)

90Brand G (porous S type 30 µm)

80
70
60
50

High DBC of BioPro IEX media is maintainedeven at higher ﬂow rate, making them suitable
for the high-speed puriﬁcation with 2-3 timesof conventional ﬂow rates. This feature oﬀers
signiﬁcant improvement on productivity.

40
30
20
10

0

200

400

600

Linear velocity (cm/hr)

9

Column: 50 X 5.0 mmI.D.
Equilibration buﬀer : 20 mM citric acid-NaOH (pH 5.3)
Elution buﬀer: 0.5 M NaCl in equilibration buﬀer
Flow rate: 200-800 cm/hr (0.66-2.62 mL/min)
Temperature: ambient (25℃)
Detection: UV at 280 nm
Sample: 1.5 mg/mL human polyclonal IgG
in equilibration buﬀer

800

1000

Excellent durability
Stability on CIP
Test protocols

Cleaning in place (CIP) is an important procedure forcleaning and sterilization of columns used for protein
puriﬁcation. The DBC and the selectivity of proteins
are unaﬀected following 20 cycles of CIP with 1 M
NaOH. The high chemical stability of BioPro IEX
media allow eﬀective cleaning with alkaline solution.

Separation of standard proteins
Determination of DBC and recovery
CIP with 1 M NaOH

DBC and recovery（5 column volumes）

120

200

DBC

100

Recovery

150

80

100

60

50

2

4

6

8

10

12

14

16

18

Number of CIP with 1 M NaOH

20

Recovery (%)

DBC (mg/mL-resin)

250

Conditions of DBC measurement

Column: BioPro IEX S75, 50 X 5.0 mmI.D.
Flow rate: 800 cm/hr (2.62 mL/min)
Equilibration buﬀer : 20 mM Glycine-NaOH (pH 9.0)
Elution buﬀer: 0.5 M NaCl in equilibration buﬀer
Sample: 1.0 mg/mL Lysozyme in equilibration buﬀer
Temperature: ambient
Detection: UV at 300 nm※DBC was determined at 10% breakthrough40

Separation of standard proteins
3 Number of CIP

2

1

20th

10th

Conditions of separation of standard proteins
ColumnEluentinitial
10

20

30

40

50 min: BioPro IEX S75, 50 X 5.0 mmI.D.
: A) 20 mM NaH2PO4-Na2HPO4 (pH 6.8)
B) 20 mM NaH2PO4-Na2HPO4 (pH 6.8) containing 0.5 M NaCl
: 0-100%B (0-60 min; Linear)
Gradient: 180 cm/hr (0.59 mL/min)
Flow rate
Temperature : 25℃: UV at 220 nm
Detection: 24 μL
Injection: 1. Ribonuclease A, 2. Cytochrome , 3. Lysozyme (0.5 mg/mL)
SampleStability on storage in alkaline solution
Change in DBC
200

DBC (mg/mL-resin)

BioPro IEX media have highstability under alkaline condition.
This feature is eﬀective for storing
the medium in alkaline solution as
well as CIP.
We recommend storing the
medium in 20% ethanol aqueous
solution in general.

160

Storage in alkaline solution(0.025 M NaOH, 25°
C)

Column: BioPro IEX Q75, 50 X 4.6 mmI.D.
Equilibration buﬀer : 20 mM Tris-HCl (pH 8.6)
Elution buﬀer: 0.5 M NaCl in equilibration buﬀer
Flow rate: 180 cm/hr (0.50 mL/min)
Sample: 1.5 mg/mL BSA in equilibration buﬀer
Temperature: 25℃
Detection: UV at 280 nm※DBC was determined at 10% breakthrough

80
40
0

Test protocols

Conditions of DBC measurement

120

0

50

100

days

150

200

Separation of standard proteins

7, 15, 30, 60,
90, 180 days

1

2

Separation of standard proteinsafter
180 days
Conditions of separation of standard proteins
ColumnEluentDetermination of DBC
initial

0

10

20

30

40

50 min: BioPro IEX Q75, 50 X 4.6 mmI.D.
: A) 20 mM Tris-HCl (pH 8.1)
B) 20 mM Tris-HCl (pH 8.1) containing 0.5 M NaCl
Gradient: 10-80%B (0-60 min; Linear)
Flow rate: 180 cm/hr (0.50 mL/min)
Temperature : 25℃
Detection: UV at 220 nm
Injection: 20 μL: 1. Transferrin (0.25 mg/mL),
Sample2. Trypsin inhibitor (0.5 mg/mL)

10

Excellent batch-to-batch reproducibility of DBC
ColumnFlow rate: 50 X 4.6 mml.D: 180 cm/hr

for anion-exchange resin
Equilibration buﬀer : 20 mM Tris-HCI (pH 8.6)
: 0.5 M NaCl in equilibration buﬀer
Elution buﬀer: 1.5 mg/mL BSA in equilibration buﬀer
Sample: UV at 280 nm
Detection

BioPro IEX S75
BioPro IEX Q75
Batch A

Batch B

Batch C

Batch Dfor cation-exchange resin
Equilibraiton buﬀer : 20 mM Glycine-NaOH (pH 9.0)
: 0.5 M NaCl in equilibration buﬀer
Elution buﬀer: 1.5 mg/mL Lysozyme in equilibration buﬀer
Sample: UV at 300 nm
Detection

Batch E

BioPro IEX media exhibit excellent batch-to-batch reproducibility of DBC. All the medium batches are inspectedby various quality control tests. We supply stable products over a long period of time.

BioPro Ion Exchange Screening Kit
The BioPro Ion Exchange Screening Kit is aset of screening columns packed with BioPro
IEX media.
・Two column types (1 mL and 5 mL) ideal for
media screening, development of puriﬁcation
methods, and loadability studies
・Easy installation and convenient use

Ordering information
BioPro IEX SmartSep Q/SPacking material

Particle
size (μm)
10BioPro IEX SmartSep Q10
BioPro IEX SmartSep S10
BioPro IEX SmartSep Q20
BioPro IEX SmartSep S20
BioPro IEX SmartSep Q30
BioPro IEX SmartSep S30

20
30

Charged group

Product number+ーR-N（CH
3）
3
ーR-SO3+3）
3
ーR-N（CHーR-SO3+3ーR-N（CH3）
ーR-SO3-QSA0S10SSA0S10QSA0S20
SSA0S20QSA0S30
SSA0S30

Charged group

Product number+ーR-N（CH
3）
3
ーR-SO3-QAA0S75
SPA0S75

BioPro IEX Q/SPacking material

Particle
size (μm)
75

BioPro IEX Q75
BioPro IEX S75

BioPro Ion Exchange Screening Kit
BioPro IEX SmartSep Q/SPacking material
BioPro IEX SmartSep Q20

BioPro IEX Q/SColumnParticle
Speciﬁcation
volumesize (μm)

Product number

1 mL
5 mL
1 mL
5 mL
1 mL
5 mL
1 mL
5 mL

BPQSA0S20-01PK
BPQSA0S20-05PK
BPSSA0S20-01PK
BPSSA0S20-05PK
BPQSA0S30-01PK
BPQSA0S30-05PK
BPSSA0S30-01PK
BPSSA0S30-05PK

20

5/pack

BioPro IEX SmartSep S20
BioPro IEX SmartSep Q30
BioPro IEX SmartSep S30

1130

5/pack

Packing material
BioPro IEX Q75
BioPro IEX S75

ColumnParticle
Speciﬁcation
volumesize (μm)

Product number

1 mL
5 mL
1 mL
5 mL

BPQAA0S75-01PK
BPQAA0S75-05PK
BPSPA0S75-01PK
BPSPA0S75-05PK

75

5/packSize Exclusion Chromatography Column for Separation of Monoclonal Antibody (MAb)

YMC-SEC MAB

Silica based SEC column optimized for separation of MAb
Suitable for separation of aggregates and fragments of MAb
Excellent resolution and peak shapes

Features

Speciﬁcations
Base

Silica gel

Particle size (μm)

3Pore size (Å)

250Functional group

Dihydroxypropyl

Usable pH range

5 - 7.5

Molecular weight range

10,000 - 700,000

Suitable for separation of aggregates and fragments of MAb
Analysis of monoclonal antibody aggregates
mAU

mAU

40

1200
1000

30

800

20

600

10

400

0

: YMC-SEC MAB (3 μm, 250 Å)
300 X 4.6 mmI.D.
Eluent: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl
Flow rate: 0.165 mL/min
Temperature : 25ºC
Detection : UV at 280 nm
Injection: 10 μL
Sample: Humanized monoclonal antibody (5 mg/mL)

10

200
0

Column

12

14

16

18

20

22 minM180604AA
5

10

15

20

25

min

Humanized monoclonal antibody was analyzed using YMC-SEC MAB. The chromatogram shows excellent peak shape,and good separation between aggregates and a monomer peak. YMC-SEC MAB is eﬀective for characterizationand quality control of antibody therapeutics.

Analysis of monoclonal antibody fragments
mAU

Intact monoclonal antibody
Digested monoclonal antibody

1

175
150
125

4

100

1. Intact antibody

2. Fab dimer

3. Fc

4. Fab*75

Column50: YMC-SEC MAB (3 μm, 250 Å)
300 X 4.6 mmI.D.
: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl
Flow rate: 0.165 mL/min
Temperature : 25℃
Detection : UV at 280 nm
Injection: 2 μL (3 mg/mL)

3

25

2

M180802EA0
M180802EA7

0
10

12

Eluent

14

16

18

20
22
24
min
*Peak derived from papain digestive fluid

A monoclonal antibody digested by papain and an intact monoclonal antibody were analyzed using YMC-SEC
MAB column. Since the column shows excellent separation between Fc and Fab fragments, which have similar
amount of MW, it is eﬀective for separation of antibody fragments.

12Batch-to-batch reproducibility

ColumnmAU

40

M180604BA2

Batch. A

M180525AA10

Batch. B

M180604AA

Batch. C

30
20
10
0

10

12

14

16

18

20

22

: YMC-SEC MAB (3 μm, 250 Å)
300 X 4.6 mmI.D.
Eluent: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl
Flow rate: 0.165 mL/min
Temperature : 25ºC
Detection : UV at 280 nm
Injection: 10 μL
Sample: Humanized monoclonal antibody (5 mg/mL)

A three-batch comparison is shown on the left.
YMC-SEC MAB achieves excellent
reproducibility of separation of monomer andaggregates as well as monomer and fragments
and is eﬀective for quality control.

min

25
20
15
10
5
0
-5

M180717AJ50

500th

M180717AH50

400th

M180717AF50

300th

M180717AD50

200th

M180717AB50

100th

M180717AA01

1st

0

2

4

6

8

10

9.5

105%

9

100%

8.5

95%

8

90%

7.5

0

100

200

300

400

500

% of initial theoreticalplate number

mAU

Elution time (min)Excellent durability

85%

Run number
Column: YMC-SEC MAB (3 μm, 250 Å)
300 X 4.6 mmI.D.
: 0.1 M NaH2PO4-Na2HPO4 (pH 7.0)
containing 0.2 M NaCl
Flow rate: 0.33 mL/min
Temperature : 25℃
Detection : UV at 280 nm
Injection: 2 μL
Sample: Humanized monoclonal antibody (1 mg/mL)
Eluentmin

YMC-SEC MAB column has excellent repeatability of separation without any change of theoretical plate number
and elution time even after repeated separation of monoclonal antibody for more than 500 times because of its
superior durability.

Ordering information
Column size
Particle
size (µm) inner diameter X length (mm)3

4.6 X 300

Please contact us about the product with other sizes.

13

Product number
DLM25S03-3046WT

Silica-based Size Exclusion Chromatography Columns

YMC-Pack Diol

Silica-based columns with high mechanical stability
Useful for molecular weight determination of proteins and sugars
High-resolution and fast analysis using 2 μm, 3 μm particle

Features

Speciﬁcations
ColumnBaseFunctional group

Pore size Particle size Usable(Å）（μm） pH range

Diol-60

60

Diol-120
Diol-200

5-7.5

200

Diol-300

For molecular weights below 10,000

3, 5

120

Dihydroxypropyl

Silica gel

2, 3, 5300

Characteristics

For molecular weights 1,000 to 100,000
For molecular weights 5,000 to 300,000
For molecular weights 20,000 to 1,000,000

Calibration curves of various proteins
Comparison of diﬀerent pore sizes (5 μm)
106Comparison of diﬀerent particle sizes (Diol-300)
1. IgM
2. Thyroglobulin
3. IgA
4. Fibrinogen
5.γ-Globulin
6. IgG
7. Transferrin
8. HSA (human serum albumin)
9.α1-Antitrypsin
10. Ovalbumin
11. Carbonic anhydrase
12. Trypsin inhibitor
13. Myoglobin14.α-Lactalbumin
15. Ribonuclease A
16. Cytochrome c

106

1
2

5 μm

3
4

3 μm
2 μm
Diol-300
MW

MW

5
6

105

Diol-200
Diol-120

7
8
9

105

10
11
12
14

104
5

7

13
15
16

9

11

104
2.0

13

2.5

3.0

Elution volume (mL)

3.5

MW
900,000
670,000
390,000
340,000
158,000
150,000
75,00066,00050,00045,00030,00020,10017,00014,10013,70012,400

4.0

Elution volume (mL)

ColumnColumnEluentEluent: YMC-Pack Diol
300 X 8.0 mmI.D.
: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl
Flow rate: 0.5 mL/min
Temperature : 25˚C
Detection: UV at 280 nm: YMC-Pack Diol
300 X 4.6 mmI.D.
: 0.1 M KH2PO4-K2HPO4 (pH 7.0)
containing 0.2 M NaCl
Flow rate: 0.165 mL/min
Temperature : 25℃
Detection: UV at 280 nm

Diol-120, Diol-200, and Diol-300 are suitable for the separation or molecular weight determination of proteins withmolecular weights from 1,000 to 1,000,000. Calibration curves of proteins with diﬀerent particle sizes using
YMC-Pack Diol are almost the same and this enables easy method transfer between UHPLC and HPLC.

Separation for standard protein markers
1

Diol-120
4

Diol-200

Diol-300
1 35
24

5

1

23

2
3

4

1. Glutamate dehydrogenase
2. Lactate dehydrogenase
3. Enolase
4. Adenylate kinase
5. Cytochrome cMW
290,000
142,000
67,00032,00012,4005
Column: YMC-Pack Diol
500 X 8.0 mmI.D.
4
7.0）
Eluent: 0.1 M KH2PO4-K2HPO（pHcontaining 0.2 M NaCl
Flow rate: 0.7 mL/min
Temperature : ambient
Detection: UV at 280 nm

0

10

20

30

min

0

10

20

30

40 min

0

10

20

30

40 minFor molecular weights from
5,000 to 300,000 substances,
Diol-200 is suitable for thisseparation.

14

Separation of peptides with molecular weights less than 10,000

2

1

3

MW
5,700
1,672
1,040
75

1. Insulin (Bovine)
2. Neurotensin
3. Angiotensin Ⅱ
4. Glycine

Diol-60
4

1

1. Myoglobin
2. Ribonuclease A
3. Cytochrome c
4. Insulin (Bovine)
5. Insulin B chain
6. α-Mating factor
7. Neurotensin
8. AngiotensinⅠ
9. CCK-Octapeptide
10. Bradykinin
11. Angiotensin Ⅱ
12. Oxytocin
13. Angiotensin Ⅲ
14. Met-Enkephalin
15. Leu-Enkephalin
16. Tetraglycine
17. Glycine

2

104

3
4
5
7
9

103
MW

Column

0

10

20

30

40 min12
14
15: YMC-Pack Diol-60
500 X 8.0 mmI.D.
Eluent: 0.1 M KH2PO4-K2HPO4（pH 7.0）
containing 0.2 M NaCl/
acetonitrile（70/30）Flow rate: 0.7 mL/min
Temperature : ambient
Detection: UV at 215 nm

6
8
11

13

10

16

102

17

10

For peptides with molecular weights less than 10,000,
Diol-60 is suitable for the separation.

15
Elution volume (mL)MW
17,00013,70012,400
5,700
3,496
1,684
1,672
1,296
1,143
1,060
1,040
1,007
931
574
556
246
75

20

Separation of oligo- and polysaccharides
Diol-200
1

1. P-800
2. P-50
3. P-20
4. P-5106

105

1
Diol-300

2
3
4
6

2

104

Dio

l-20

7

0

8
9

103

Diol-60

10

Dio

l-1

11

12
13

102

10
0

MW
1. Pullulan (P-800)
853,000
2. Pullulan (P-400)
380,000
3. Pullulan (P-200)
186,000
4. Pullulan (P-100)
100,000
5. Pullulan (P-50)
48,000
6. Pullulan (P-20)
23,700
7. Pullulan (P-10)
12,2008. Pullulan (P-5)
5,800
9. Maltopentadecaose (G15)2,448
10. Maltoundecaose (G11)1,800
11. Maltoheptaose (G7)1,152
12. Maltopentaose (G5)824
13. Maltotriose (G3)504
14. Maltose (G2)342
15. Glucose (G1)
180

5

4
MW

3

MW
853,000
48,00023,7005,800

10

20 min

20

14
15

15
Elution volume (mL)20

25

For separation or molecular weight determination of water-soluble
oligo- and polysaccharides, Diol-60, Diol-120, Diol-200, and Diol-300
are useful individually or in combination.

Column: YMC-Pack Diol, 500 X 8.0 mmI.D.
Eluent: water
Flow rate: 1.0 mL/min
Temperature : ambient
Detection: RI

Ordering information
YMC-Pack Diol (stainless steel column)

Packing
material

Particle
size (μm)

PoreSize (Å)

Column size
inner diameter X length (mm)Product number

Packing
material

Particle
size (μm)

PoreSize (Å)

Column size
inner diameter X length (mm)Product number

Diol-60

360

4.6 X 300

DL06S03-3046WT

Diol-2002

200

4.6 X 150

DL20S02-1546PTH

560

4.6 X 300

DL06S05-3046WT

4.6 X 300

DL20S02-3046PTH

8.0 X 300

DL06S05-3008WT

3200

4.6 X 300

DL20S03-3046WT

8.0 X 500

DL06S05-5008WT

5200

4.6 X 300

DL20S05-3046WT

20 X 300

DL06S05-3020WT

8.0 X 300

DL20S05-3008WT

20 X 500

DL06S05-5020WT

8.0 X 500

DL20S05-5008WT

Diol-1203

120

4.6 X 300

DL12S03-3046WT

20 X 300

DL20S05-3020WT

5120

4.6 X 300

DL12S05-3046WT

20 X 500

DL20S05-5020WT

8.0 X 300

DL12S05-3008WT

4.6 X 150

DL30S02-1546PTH

8.0 X 500

DL12S05-5008WT

4.6 X 300

DL30S02-3046PTH

20 X 300

DL12S05-3020WT

3300

4.6 X 300

DL30S03-3046WT

20 X 500

DL12S05-5020WT

5300

4.6 X 300

DL30S05-3046WT

8.0 X 300

DL30S05-3008WT

8.0 X 500

DL30S05-5008WT

20 X 300

DL30S05-3020WT

20 X 500

DL30S05-5020WT

2300

Please see our website for guard columns and glass columns.

Consideration of connector and column ﬁttings
Tubing and connector

Diol-300

ColumnThe end of the product number＊Port depth

Style of endﬁtting

PTH

2 mm

Parker style (UPLC compatible)

WT

3 mm

Waters style

UPLC is registered trademark of Waters Corporation＊Port depth15

Hydrophobic Interaction Chromatography Column

BioPro HIC BF

Eﬀective for separation of proteins and antibodies such as Antibody-Drug Conjugates (ADCs)
High resolution comparable to sub-3 μm with low operating pressure
Fast analysis at high ﬂow rates
Usable for laboratory-scale puriﬁcation

Features

Speciﬁcations
MatrixHydrophilic non-porous polymer

Particle size (μm)

4Functional group

Butyl group

Usable temperature

10- 60℃

Usable pH range

2‒12

Eﬀective for Drug-to-Antibody Ratio (DAR) analysis of ADCs
ColumnEluentmV
50

Flow rate
Temperature
Detection
Sample40
Strong conjugation
condition

30*Courtesy by RIKEN.

L180515B

20

ADCs with diﬀerent conjugation condition wereanalyzed using BioPro HIC BF. These results indicate
the increase of ADC with high DAR under thestrong condition compared to the weak condition.
BioPro HIC BF column provides superior separation
for ADCs, therefore it is eﬀective for DAR
determination and monitoring of conjugation reactions.

Weak conjugation
condition

10

L180515A

Unconjugatedantibody

0

L180515C

5

0

10

15

20

25

: BioPro HIC BF 4 μm, 100 X 4.6 mmI.D.
: A) 50 mM NaH2PO4-Na2HPO4 (pH 7.0) containing
1.5 M (NH4)2SO4/2-propanol (95/5)
B) 50 mM NaH2PO4-Na2HPO4 (pH 7.0)/2-propanol (80/20)
0%B (0-1 min), 0-100%B (1-15 min), 100%B (15-20 min),
0%B (20-30 min)
: 1.0 mL/min: 25℃: UV at 280 nm: Antibody Drug Conjugate*

minHigh resolution comparable to sub-3 μm with low operating pressure
BioPro HIC BF (4 µm)

mAU
120

Competitorʼs HIC column (2.5 µm)

mAU
120

3.3-4.6 MPa

100
80

80

Rs (1,2)= 4.57
Rs (2,3)= 3.49
Rs (3,4)= 11.17

60
40

Rs (1,2) = 2.78
Rs (2,3) = 4.37
Rs (3,4) = 10.87

60
40

20
0

7.3-10.5 MPa

100

20
L171208D02

0.0

2.5

0
5.0

7.5

10.0

12.5

15.0

ColumnEluent17.5: 100 X 4.6 mmI.D.
: A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4
B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0)0-100%B (0-11 min), 100%B (11-15 min)Flow rate: 0.5 mL/min
Temperature : 25℃
Detection: UV at 280 nm
Injection: 15 μLmin

L171212D02

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

min

The separation of proteins is compared on BioPro
HIC BF with particle size 4 μm and a competitorʼs
HIC column with particle size 2.5 μm. BioPro HIC
BF with optimized media design and packing
technology has superior resolution comparable tocompetitorʼs column with sub-3 μm.

Ordering information
Particle size
(μm)

Column size
inner diameter X length (mm)Product number

4

4.6 X 100

BHB00S04-1046WT

Please contact us about the product with other sizes. Preparative columns are also available.

16

Reversed-phase Chromatography Columns

YMC-Triart/YMC-Triart Bio
Features

Useful for separation of biomolecules such as peptides, proteins,antibodies, and oligonucleotides
Excellent chemical durability enables analysis under high temperature
Good peak shapes with mobile phase suitable for LC/MS
Various column sizes are available for separation from analysis
to preparative scale

Speciﬁcations
Triart C18

Triart C8

Triart Bio C4

Base

Organic/inorganic hybrid silica

Particle size (μm)

1.9, 3, 5
120

Pore size (Å)

300

Bonding

Trifunctional

Endcapping

Yes

Usable pH range

1-12

1-10

Max. temperature

90℃ for pH 1-7
50℃ for pH 7-12

90℃ for pH 1-7
50℃ for pH 7-10

Please see our website for the columns with other functional groups.

Versatile hybrid base material
YMC-Triart is based on novel organic/inorganic hybrid particles.
The particle combines high mechanical stability and high eﬃciency
derived from silica based packing material and high chemical
stability derived from polymer based packing material. The
granulation process utilizing microreactor technology enables
continuous and highly controlled production of hybrid particles. The
particle has uniform pore size distribution and smooth surface aswell as uniform particle size. This feature greatly contributes to
excellent peak shape and separation reproducibility.

Excellent chemical durability
pH 1 (1% TFA), 90℃

Test conditions% of initial retention

Column17

Triart C18

100
90

Triart Bio C480
70

Silica based wide pore C460

The columns are kept in acetonitrile/water/
TFA(10/90/1, pH 1) at 90℃, and tested
for performance every 20 hours

50
40: 5 μm, 150 X 3.0 mmI.D. for C4
5 μm, 50 X 2.0 mmI.D. for C18
Eluent: acetonitrile/water (60/40)
Flow rate: 0.4 mL/min for 3.0 mmI.D.
0.2 mL/min for 2.0 mmI.D.
Temperature : 37℃
Sample: butyl benzoate

0

20

40

60

Time (hr)80

100Triart columns are eﬀective in improving
chromatographic outcomes for stabilizing retention time(and also peak shape) due to the use of innovative
surface modiﬁcation of the organic/inorganic hybrid
silica. When separating proteins and peptides, Triart
oﬀers excellent durability even when using mobile
phases containing up to 1% TFA or column temperaturesof 90℃ are utilized.

Molecular weight of sample

Column selection guide for proteins and peptides
Columns are selected based on molecular weights ofthe target substances to separate proteins andpeptides. Triart C18/C8 with pore size 120 Å provide
the good separation of substances with molecularweight of up to 30,000 under high temperature. For
separation of substances with larger molecules, wide
pore columns are eﬀective. Triart Bio C4 with poresize 300 Å can perform separation of substanceswith molecular weight of up to 150,000 under hightemperature.
An elevated temperature can improve eﬃciency andpeak shape by lowering the viscosity of the mobile
phase and improving mass transfer. The appropriate
molecular weight range for a given pore size of Triart
can be expanded compared to using the same poresize at lower temperature.

Triart C18
Triart C8(120 Å)30,000
Triart Bio C4(300 Å)150,000

Eﬀect of column temperature on separation1

Rs (3,4) = W1/2 (1) = 0.019
W1/2 (5) = 0.046

40℃

2
3, 4
5

F120330H

Rs (3,4)

50℃

=

1

1.97

1. Oxytocin
2. Leu-Enkephalin
3. β-Endorphin
4. Insulin
5. β-Lactoglobulin A

2
3

W1/2 (1) = 0.017
W1/2 (5) = 0.039

45F120330J

Column1

3

4

5

W1/2 (5) = 0.034

F120330K

1

Rs (3,4) = 4.87
W1/2 (1) = 0.015
W1/2 (5) = 0.030

70℃
F120330L

0

0.5

Increasing column temperature to
70℃ provides selectivity change,
sharper peaks, and therefore
improved resolution especially forlarger molecules.

2
3

1: YMC-Triart C18 (1.9 μm, 120 Å)
50 X 2.0 mmI.D.
Eluent: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
10-80%B (0-5 min)Flow rate : 0.4 mL/min
Detection : UV at 220 nm

2

Rs (3,4) = 3.47
W1/2 (1) = 0.016

60℃(MW 1,007)(MW
556)(MW 3,465)(MW 5,733)(MW 18,400)

1.5

4

5

2

2.5

3

min

Useful for separation of macromolecules such as antibodies
mAU

Column1400

Eluent: YMC-Triart Bio C4 (3 μm, 300 Å)
150 X 3.0 mmI.D.
: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
30-60%B (0-15 min), 90%B (15-30 min)Flow rate : 0.4 mL/min
Detection : UV at 220 nm
Injection : 4 μL
Sample : Humanized monoclonal IgG1

Good peak shapes
at 70℃ or higher

1200
1000

Adsorption

800

Adsorption

600
400
200
0
2

4

6

8

10

V180219D2

40℃

V180219G2

50℃

V180219K2

60℃

V180219N2

70℃

V180219R2

80℃

V180219U2

90℃

min#

Intact monoclonal antibody was
analyzed at temperatures between 40℃
and 90℃ using Triart Bio C4. Good peak
shapes were acquired at 70℃ andhigher while there was no elution at 50℃
and lower. Highly durable Triart Bio C4
can perform stable analysis even at 90℃
and is suitable for the reversed-phaseanalysis of antibodies at hightemperatures.

18

Good peak shapes with mobile phase suitable for LC/MS
Competitorʼs C4 column(1.7 μm, 300 Å)

Triart Bio C4 (1.9 µm, 300 Å)Competitorʼs core-shell C4 column(3.5 μm, 450 Å)

mAU
20

mAU
20

mAU
20

15

15

15

10

10

10

5

5

5

0

0
0

1

2

0

V180706H
V180706K2

V180705H2
V180705B2

3

4

5

6

7

8

9

min

0

#

1. Fc/2
2. Light chain
3. Fd’

1

2

3

4

5

6

7

8

9 min

#

V180709B
V180709D1

0

1

2

3

4

5

6

7

8

9

min#

Mobile phase containing TFA
Mobile phase containing formic acid

【Sample】

F(ab’)2Fd’

IdeS
digestion

Denaturation
reduction
Fc/2

Fc/2

Column size
Eluent <TFA>

Light chain: 150 X 2.1 mmI.D.
: A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.1)
25-50%B (0-10 min), 90%B (10-12.5 min)Eluent <Formic acid> : A) water/formic acid (100/0.1)
B) acetonitrile/formic acid (100/0.1)
20-45%B (0-10 min), 90%B (10-12.5 min)Detection: UV at 280 nm
Flow rate: 0.4 mL/min
Temperature: 80℃
Injection: 4 μL (0.25 mg/mL)
Sample: mAb Subunit Standard (Waters Corporation)

Above are the chromatograms of monoclonal antibody fragments
analysis with mobile phase containing either TFA or formic acid.
Triart Bio C4 achieves excellent peak shape even when using mobile
phase containing formic acid.
Therefore it is ideal for the high sensitive LC/MS analysis.

Peptide mapping
PC = 450

100 X 2.0 mmI.D.
1.9 µm
27.6-28.6 MPa(4000-4150 psi)

P110728A

1

12 minColumnEluentPC = 626

200 X 2.0 mmI.D.
1.9 µm(Two coupled 1.9 μm,
100 X 2.0 mmI.D.)
58.1-61.6 MPa(8430-8930 psi)

2

P110707A

24 min：YMC-Triart C18 (1.9 μm, 120 Å)：A) water/TFA (100/0.1)
B) acetonitrile/TFA (100/0.08)
5-40%B (0-15 min) for a single column
5-40%B (0-30 min) for two coupled columns
Flow rate
0.4 mL/min
Temperature：70℃
Detection ：UV at 220 nm
Injection：10 μL for a single column
20 μL for two coupled columns
Sample：Triptic digest of Bovine Hemoglobin
System：Agilent 1290

Triart 1.9 μm column has superior column eﬃciency, and a coupling of two 100 mm length of Triart 1.9 μm columns
oﬀers outstanding separation ability. This allows the precise separation in an analysis of complicated samples, suchas peptide mapping.

19

Separation of oligonucleotides
Column : YMC-Triart C18 (5 μm, 120 Å)
150 X 2.0 mmI.D.
Flow rate : 0.2 mL/min
Gradient : 35-50%B (0-84 min)

mAU
20
15

Oligonucleotides d(T) 2-20

10

Eluent: A) 10 mM di- -butylamine-acetic acid (pH 6.0)
B) methanol
Temperature : 35℃
Detection: UV at 269 nm
Injection: 1 μL (5 nmol/mL)

500F111116H

10

20

30

40

50

60

80

70

min

Column : YMC-Triart C18 (1.9 μm, 120 Å)
50 X 2.0 mmI.D.
Flow rate : 0.8 mL/min
Gradient : 35-50%B (0-7 min)

mAU
20
15
10
5
0
0

1

F111109K

2

3

4

5

6

7 min

In the separation of oligonucleotides, 19 peaks
are completely resolved within 7 minutes usinga Triart C18 1.9 µm UHPLC column. The
separation is achieved within one tenth of theanalysis time of the conventional HPLC
method.

Ordering information
Packing material

Code

Column length (mm)Code

Inner diameter (mm)Triart C18

TA

20

02

2.0

02

Triart C8

TO

30

03

2.1

Q1

Triart Bio C4

TB

33

H3

3.0

03

35

H5

4.0

04

50

05

4.6

46

75

L5

6.0

06

Code

Pore size (Å)

Code

120

12

100

10

10

10

300

30

125

R5

20

20

150

15

30

30

250

25
Column Type

Code

Particle size (μm)

Code

1.9

SP9

3

S03

5

S05

Waters style

WT/WX

Parker style

PT/PTH

Metal free column

PTP

Example) YMC-Triart C18 1.9 μm, 100 X 2.0 mmI.D.
Packing material Pore size Particle size Column length Inner diameter Column Type
TA
12
SP9
10
02
PT

Product number：TA12SP9-1002PT

Consideration of connector
and column ﬁttings
Tubing and connector

ColumnThe end of the product number＊Port depth

Style of endﬁtting

PT / PTH / PTP

2 mm

Parker style (UPLC compatible)

WT / WX

3 mm

Waters style

UPLC is registered trademark of Waters Corporation＊Port depth

Please note that combinations of features cannot be selected at random, but only from the possible speciﬁcations for a chosen stationary phase.
These can be determined from our website.

20

Preparative Systems/Columns

Glass Columns

ECOPLUS
Features

Bio compatible
Universal application
Aqueous buﬀer (AB) versions and solvent resistant (SR) versions
are available
Low temperature versions available with polyethylene plunger
and EPDM sealing ring
Height adjustable plungers at both ends
Easy to use
Compatible with any LC systems

Versatile high-performance glass columns
ECO PLUS glass columns are multi-purpose columns for almost all types ofliquid chromatography applications (pressure limit 1.5 to 8.0 MPa). They areavailable in two forms; AB (Aqueous Buﬀer) versions for aqueous buﬀer andcold room applications, and SR (Solvent Resistant) versions for separations
involving organic solvents.
With a choice of adjustable length plungers at each ends and a totally inert
triple chevron sealing system, the SR versions support the complete
spectrum of applications from classical normal phase and reversed-phase
chromatography through to biochromatography.
The “Quick-Lock” connection system makes it possible to open and seal
the column simply and quickly.

“Quick-Lock” Fitting

Easy self packing with optional packing adaptors
Packing adapters are used if the packing methods requires additional
column volume during the packing process. More volume than given by thecolumn is required when the column is packed with the slurry method. The
ECO PLUS series of packing adapters consist of a column coupler and an
empty glass body. These must be of the same diameter as the column tobe packed and must be used as packing adapters, not for extending thelength of a column body during use.

Speciﬁcations
Model
Inner diameter (mm)Pressure limit
(MPa)

TAC05

TAC10

TAC15

TAC25

TAC35

TAC50

5

10

15

25

35

50

AB

8.0

8.0

7.0

5.0

4.0

3.0

SR

8.0

5.0

5.0

5.0

4.0

1.5

Column lengths (mm)Usable temperature
range

125, 250, 500
AB

4 ‒ 40℃

SR

16 ‒ 40℃
1/4”‒28G ﬁttings（1/16” tubing）

Connection
Frit

AB
SR

Options
Please see our website for more details

21

1/4”‒ 28G ﬁttings（1/8” tubing）
Polyethylene

Sintered glass

Stainless steel

Short plungers, short/long plungers, long plungers

Biochromatography Systems/Columns
Features

Suitable for downstream puriﬁcation process for biophamaceuticals

Preparative LC systems BioStream
Compliance with cGMP
Sanitary design superior in cleaning
Maximum ﬂow rate: 30 L/min
Customization is available upon request

Glass columns YMC Pilot
All wetted parts are made of non-metal
Safe and easy handling for packing
Wide selection of inner diameter
Various options available

Glass columns YMC Pilot PLUS
Upgraded columns of YMC Pilot
Signiﬁcant improvement for handlingSpeciﬁcations
BioStream
Model

BSTP-800

BSTP-03K
BSTS-03K

BSTS-10K

BSTS-30K

800

3000

10000

30000

1300 × 1200 × 1800

1800 × 1500 × 1800

Max. ﬂow rate (mL/min)
System pressure limit (MPa)

0.5 (Max. 0.6)

Dimensions (mm) (W x D x H)

800 × 900 × 1300

900 × 1100 × 1800

Control software

BioStream software

GMP/CSV support

Yes

Explosion-proof

No

Features

Sanitary design, 3 wavelengths measurable simultaneously

YMC Pilot
Model
Inner diameter (mm)PI100/500
PI100/850

PI140/500
PI140/850

PI200/500
PI200/850

PI300/500
PI300/850

100

140

200

300

500, 850

Column lengths (mm)Pressure limit (MPa)1.0500, 850
0.7

0.5

0.3

Please contact us for the information of YMC Pilot PLUS

22

Contract Puriﬁcation

From Laboratory to Plant-scale
Separation/Puriﬁcation
Features

Chromatographic puriﬁcation with preparative LC systems
manufactured in-house
Puriﬁcation method development to meet various requirements
Seamless scale-up depending on the development stages
Smooth transfer of the puriﬁcation method and system to clients

Advantages of contract puriﬁcation
Problems for customers

The solutions by outsourcing
to YMCHigh Purity
Compounds

Diﬃculty in puriﬁcation method

Accumulated experience and know-

optimization

how of separation and puriﬁcation

Quick Service

Expensive columns and packing

Low-cost columns and packing

materials for puriﬁcationmaterials manufactured in-house

No systems and facilities forLarge columns and preparative

preparative puriﬁcation

systems are available

High Reliability
Low-Cost

Shortage of time and labor

Applicable Scale

Target Substances

GMP： grams to tons
non-GMP ： milligrams to tons

Oligonucleotides
PeptidesProteins
Chiral compounds

Before use (installation, operation, maintenance or check-up) of our products, instruction manuals should be carefully read and understood, and safety rules and precautions followed as outlined in the manuals.

Worldwide Availability
YMC America, Inc.

YMC Europe GmbHYMC Switzerland LLC

YMC Shanghai Rep. Officewww.ymcamerica.com

www.ymc.de

www.ymc-schweiz.ch

www.ymcchina.com

YMC India Pvt. Ltd.

YMC Korea Co., Ltd.

YMC Taiwan Co., Ltd.

YMC Singapore Tradelinks Pte. Ltd.

www.ymcindia.com

www.ymckorea.com

www.ymctaiwan.com

www.ymc.sg

Distributor

YMC Karasuma-Gojo Bldg., 284 Daigo-cho
Karasuma Nishiiru Gojo-dori, Shimogyo-ku
Kyoto, 600-8106, Japan
TEL:+81-75-342-4515 FAX:+81-75-342-4550
www.ymc.co.jp sales@ymc.co.jp
18111K A E
The above information is as of November, 2018 and is subject to change without notice. ©2018 YMC CO., LTD. All Rights Reserved.