www.ymc.co.jphttp://www.ymc.co.jp

A newly developed hydrophilic polymer-based ion exchange chromatography media and

purification of Immunoglobulin Y from egg yolkErnest J. Sobkow*1, Noriko Shoji*2, Akiko Matsui*2, Saoko Nozawa*2, Taeko Nakajima*2, Masakatsu Omote*2 and Naohiro Kuriyama*2

YMC America, Inc.*1, YMC Co., Ltd.*2

Introduction

The recent development of bio-pharmaceutical industry has been remarkable, and shortening the development

time and reducing the cost become increasingly important. The development of efficient, economical and

selective separation method is required for successful commercialization of bio-pharmaceutical products. To

meet these demands, we have developed new polymeric packing materials named YMC-BioPro series, which

are specially designed for ion exchange (IEX) separation and purification of proteins, peptides and nucleic

acids. YMC-BioPro series includes the packed columns with 5 micron porous/non-porous polymer for analysis

and laboratory scale purification, and the bulk materials of 30, 75 micron porous polymer for capture and

intermediate purification. The all materials are based on the same hydrophilic polymer beads with low

nonspecific adsorption.

In this poster, we show the properties and benefits of these new IEX chromatography media, including

separation selectivity of some important biomolecules such as monoclonal antibody and DNA, binding capacity

and recovery at various linear velocity, and chemical stability under cleaning-in-place condition with 1.0 M

NaOH. We also present the efficient purification protocol of Immunoglobulin Y (IgY) from egg yolk.

Features of new IEX chromatography media

2～～～～122～～～～12Available pH range

Q30/Q75 : >160 (BSA)

S30/S75 : > 160 (Lysozyme)

QA : >110 (BSA)

SP > 70 (IgG)

QA-F : >12 (BSA)

SP-F : >10 (IgG)

Dynamic binding capacity

(mg/ml-resin)

Q30/Q75 : -CH2N+(CH3)3

S30/S75 : -(CH2)4SO3-

QA-F/QA : -CH2N+(CH3)3

SP-F/SP : -(CH2)3SO3-

Charged group

porous polymer beadsporous polymer beadsnon-porous polymer beadsMatrix

75305Particle size (µµµµm)

YMC-BioPro Q75/S75YMC-BioPro Q30/S30YMC-BioPro QA/SPYMC-BioPro QA-F/SP-F

Bulk materialsPre-Packed Columns

� Newly developed hydrophilic polymer with low nonspecific adsorption

� Porous polymer beads with high binding capacity and high recovery of biomolecules

� Non-porous polymer beads with high chemical and mechanical stabilities

� 5 µm packed column for high-resolution and high-throughput analysis, or laboratory-scale purification

� 30, 75 µm porous bulk materials for capture and intermediate purification

Comparison of selectivity and resolution on non-porous/porous IEX

columns

Eluent : A) 20 mM Tris-HCl (pH 8.6)

B) 20 mM Tris-HCl (pH 8.6 containing 0.5 M NaCl

0-30%B (0-15 min), 30-100%B (15-30 min)

Flow rate : 0.5 ml/min

Temperature : 25℃

Detection : UV at 280 nm

Injection : 20 µl

Sample : Human serum (100 µl/ml)

Eluent : A) 20 mM Tris-HCl (pH 8.1)

B) 20 mM Tris-HCl (pH 8.1) containing 0.5 M NaCl

10-25%B (0-18 min)

Flow rate : 1.0 ml/min (180 cm/hr)

Temperature : 25℃

Detection : UV at 220 nm

Injection : 10 µl (0.1 mg/ml)

Sample : Monoclonal mouse IgG1 against human IgG4 (Purified by DEAE chromatography, containing NaN3)

P080219A

min0 4 8 12 16

mAU

0

2

4

MAb

NaN3

Brand T (non-porous DEAE type)

2.5 µµµµm, 35 X 4.6 mm i. d.

7.5 MPa

P080220B

min0 4 8 12 16

mAU

0

2

4

NaN3

MAb

Brand G (non-porous Q type)

3 µµµµm, 50 X 4.6 mm i. d.

5.9 MPa

min0 4 8 12 16

mAU

0

2

4

P080220A

NaN3

MAb

YMC-BioPro QA-F

5 µµµµm, 30 X 4.6 mm i. d.

2.5 MPa

Analysis of IgG1 monoclonal antibody (MAb) on non-porous type anion-exchange columns

Brand G (porous Q type)

10 µµµµm, 50 X 5.0 mm i. d.

0 10 20 30 min

0

10

mAU

Brand T (porous Q type)

10 µµµµm, 50 X 4.6 mm i. d.

0 10 20 30 min

0

10

mAU

YMC-BioPro QA

5 µµµµm, 50 X 4.6 mm i. d.

AlbuminIgG

Transferrin

0 10 20 30 min

0

10

mAU

Analysis of proteins in human serum on porous type anion-exchange columns

The separation of biological samples is compared on various commercially available IEX columns in analytical scale. As shown

in the left figures, non-porous type YMC-BioPro QA-F column can achieve superior resolution of MAb in less analysis time at

lower back pressure than two other columns which are packed with smaller particles. The right figures show that porous type

YMC-BioPro QA is suitable for high-resolution analysis of protein samples containing a large amount of impurities. The optimal

packed 5 µm spherical monodispersed beads with proprietary surface modification provides high efficiency and high selectivity

to YMC-BioPro columns.

0 5 10 15 20 min

0

50

mAU

N80221B

0 5 10 15 20 min

0

50

mAU

N80212G

Eluent : A) 20 mM KH2PO4-K2HPO4 (pH 6.8)

B) 20 mM KH2PO4-K2HPO4 (pH 6.8) containing 0.5 M NaCl

0-100%B (0-4 min) for YMC-BioPro SP-F

0-100%B (0-60 min) for YMC-BioPro SP

Temperature : 25℃

Detection : UV at 220 nmInjection : 20 µl

1. Ribonuclease A

2. Cytochrome c

3. Lysozyme

YMC-BioPro QA-F

100 X 4.6 mm i. d.

YMC-BioPro QA-F

30 X 4.6 mm i. d.

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℃

Detection : UV at 260 nm

Injection : 20 µ l (0.25 mg/ml)

High-throughput analysis on non-porous short column with higher flow

rate

Analysis of standard proteins

Porous type

YMC-BioPro SP

5 µµµµm, 50 X 4.6 mm i. d.

0

0.5

0 20 40 60 min

AU 321

0.5 ml/min

2.1-2.0 MPa

Non-porous type

YMC-BioPro SP-F

5 µµµµm, 30 X 4.6 mm i. d.

0

0.2

0.4

AU

2 4 min

1

3

2

0

1.5 ml/min

4.8-5.2 MPa

0

0.2

0.4

AU

1 2 3 4 minN071023K

1

3

2

95% degrease in analysis time

The high mechanical stability of non-porous

polymer beads enables faster elution of

proteins with higher flow rate. The 30 mm and

50 mm-length column of YMC-BioPro

QA/F/SP-F is designed for high-throughput

analysis with low operating pressure.

High-resolution analysis on non-porous 100 mm-length columnAnalysis of DNA fragment

1 kb DNA ladder (75-12,216 bp)

The separation of DNA fragments is compared

between 100 mm-length and 30 mm-length of

YMC-BioPro QA-F columns. The resolution of

DNA fragments is dramatically improved by

100 mm column.

The combination of non-porous polymer beads

and long column length provides extremely

high column efficiency.

Column size : 50 X 5.0 mm i. d.

Sample : 1.0 mg/ml Lysozyme in equilibration buffer

Equilibration buffer : 20mM Glycine-NaOH (pH 9.0)

Elution buffer : 20mM Glycine-NaOH (pH 9.0) containing 0.5 M NaCl

Detection : UV at 300 nm

Column size : 55 X 5.0 mm i. d.

Sample : 1.5 mg/ml BSA in equilibration buffer

Equilibration buffer : 20mM Tris-HCｌ (pH 8.6)

Elution buffer : 20mM Tris-HCｌ (pH 8.6) containing 0.5 M NaCl

Detection : UV at 280 nm

Comparison of dynamic binding capacity (DBC) at different flow rate on

IEX resins for capture and intermediate purification

The dependency of DBC to linear velocity

is compared on various commercially

available IEX resins for capture and

intermediate purification. YMC-BioPro

S75 and Q75 shows the high DBC over a

wide range of linear velocity, and the

difference of DBC is only less than 5%

between 200 cm/hr and 1000 cm/hr.

Use of IEX resins at flow rates of up to

1000 cm/hr at high DBC enables rapid

large scale purification. This shows 75 µm

BioPro resins would give increased

productivity and reduced cost in

biopharmaceutical production.

Cation-exchange

YMC-BioPro S75 (porous S type, 75 µm)

Brand T (porous S type, 50-100 µm)

Brand G (porous S type, 90 µm)

Anion-exchange

YMC-BioPro Q75 (porous Q type, 75 µm)

Brand T (porous Q type, 50-100 µm)

Brand G (porous Q type, 90 µm)

Initial

10 cycles

20 cycles

1 23

10 20 30 40 50 min

1. Ribonuclease A

2. Cytochrome c

3. Lysozyme

Numbers of CIP with 1.0M NaOH

120

100

60

80

250

200

100

150

DBC (mg/m

l-gel)

Recovery (%)

405010 202 4 6 8 12 14 16 18

DBC

Recovery

Column : YMC-BioPro S75, 50X5.0 mm i. d.

Conditions of DBC determination

Equilibration buffer : 20 mM Glycine-NaOH (pH 9.0)

Elution buffer : 0.5 M NaCl in equilibration buffer

Flow rate : 2.62 ml/min (800 cm/hr)

Sample : 1.0 mg/ml Lysozyme in equilibration buffer

Temperature : ambient (25℃)

Detection : UV at 300 nm

※DBC is determined at 10％ breakthrough.

Conditions of protein separationEluent : A) 20 mM NaH2PO4-Na2HPO4 (pH 6.8)

B) 20 mM NaH2PO4-Na2HPO4 (pH 6.8)

containing 0.5 M NaCl

Gradient : 0-100％B (0-60 min, Linear)

Flow rate : 0.59 ml/min (180 cm/hr)

Temperature : 25 ℃Detection : UV at 220 nm

Injection : 24 ul

Cleaning-in-place (CIP) study of BioPro S75 resin

Separation of proteins

Determination of DBC

and recovery

CIP with 1.0M NaOH

(5 CV at 400 cm/hr)

Test protocols DBC and recovery Separation of standard proteins

The DBC and the selectivity of protein separation are unaffected following 20 cycles of CIP with 1.0 M NaOH. The high

chemical stability of BioPro resins allow effective cleaning with alkaline solution. The recovery is maintained at a constant

value around 100%. These shows the hydrophilic properties of the matrix polymer remarkably reduce nonspecific

adsorption of proteins.

IgY (MW 170 kDa)

H chain

(MW 65 kDa)

L chain

(MW 23 kDa)

Sample : Crude extract from egg yolk

Column : YMC-BioPro Q75, 75 µm, 50 X 4.6 mm i. d.

Eluent : A) 20mM Tris-HCl (pH 8.1)

B) 20mM Tris-HCl (pH 8.1) containing 0.5M NaCl

10%B (0-15 min), 30%B (15-30 min), 90%B (30-40 min)

Flow rate : 0.5 ml/min (180 cm/hr)

Temperature : ambient

Detection : UV at 280 nm

Injection : 1 ml (approx. 20 mg protein)

min

mV

0 10 20 30 40 50 60

0

100

200

300

400

500

A090824C

Step 1:

Capture purification by ion-exchange chromatography

(IEC)

min

mV

0 5 10 15 20 25 30

0

25

50

75

100

A090825A

Sample : Fraction from capture purification by IEC

Column : YMC-Pack Diol-200, 5 µm, 300 X 8.0 mm i. d.

Eluent : 0.1M KH2PO4-K2HPO4 (pH 6.9) containing 0.2 M NaCl

Flow rate : 0.7 ml/min (180 cm/hr)

Temperature : ambient

Detection : UV at 280 nm

Injection : 1 ml (approx. 0.45 mg IgY)

A090826A

min0 5 10 15 20

A090824J

A090826E

Two step purification of IgY from egg yolk extract

Step 2:

Final purification by size-exclusion chromatography

(SEC)

*Crude extract from egg yolk containing IgY

YMC-BioPro Q75, 75 µµµµm

YMC-Pack Diol-200, 5 µµµµm

*Crude extract is the supernatant after precipitation of

the lipids and lipoproteins from egg yolk.

Lane

1: Marker

(containing SDS)

2: Crude extract

3: IEC fraction

4: SEC fraction

5: Standard IgY

Crude extract

IEC fraction

from step 1

SEC fraction

from step 2

Column : YMC-Pack Diol-200

5 µm, 300 X 4.6 mm i. d.

Eluent : 0.1M KH2PO4-K2HPO4 (pH 6.9)

containing 0.2 M NaCl

Flow rate : 0.23 ml/min

Temperature : ambient

Detection : UV at 280 nm

Purity <15%

Purity 53%

Recovery 40%

Purity >99%

Recovery 23%

Analysis of crude and purified fraction

97

66

45

29

20

14

MW

(kDa)

1 2 3 4 5

205

116

97

80

66

55

45

30

21

14

6.5

MW

(kDa)

1 2 3 4 5

H chain

L chain

Non-reduced SDS-PAGE SDS-PAGE

SEC analysis

Egg yolk antibody, IgY, is purified by two chromatographic steps using

IEC as a capture purification and SEC as a final purification. Purity of

the IgY is checked with SDS-PAGE and SEC analysis shown below.

This purification protocol can isolate IgY with high purity more than

99%.

Conclusions

� Using the optimal IEX material for a specific application can result in a significant decrease in the costs of

the biopharmaceutical production. YMC-BioPro series, which based on the same hydrophilic polymer with

low non-specific adsorption, is scalable from analytical to large-scale preparative separation.

� 5 µm pre-packed columns with optimal packing technology provide superior resolution. Non-porous type

BioPro QA-F/SP-F columns are effective for high resolution analysis or QC assessment of complex

mixtures, such as MAbs. Porous type BioPro QA/SP columns are useful for analysis and laboratory-scale

purification of biological samples.

� 30 µm and 75 µm bulk materials of porous polymer are useful for high capacity capture and high

efficiency intermediate purification steps.