04

Hydrophobic Interaction
Chromatography Column

BioPro HIC BF----------------------------------------- 38-39
Ordering Information------------------------------------- 39

04

Hydrophobic Interaction Chromatography Column

BioPro HIC BF
l Effective for separation of proteins and antibodies such as ADCsl High resolution comparable to sub-3 μm with low operating pressurel Usable for laboratory scale purification■■

Particle size : 4 μm
Usable pH range : 2-12

Hydrophobic interaction chromatography column for separation of biologics

Hydrophobic Interaction Chromatography Column

BioPro HIC BF is based on a non-porous hydrophilic polymer. It is designed and optimized for analysis and laboratory scale
purification of antibodies, antibody-drug conjugates (ADCs) and other proteins.
Optimized surface modification and column packing method realize high separation performance. High-throughput analysis with afaster flow rate can be achieved by the rigid particle and moderate back pressure.

Specifications
BioPro HIC BFMatrix

Hydrophilic non-porous polymer

Particle size (μm)

4Bonded phaseButyl group

Usable temperature

10-60℃

Usable pH range

2-12

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

ColumnEluentmV5040Strong conjugation
condition

30
L180515B

20Weak conjugation
condition

10Unconjugatedantibody
L180515C

038*Courtesy by RIKEN.

L180515A

0510152025: 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)Flow rate: 1.0 mL/min
Temperature : 25°CDetection: UV at 280 nm
Sample: Antibody Drug Conjugate*min

ADCs with different conjugation condition were analyzed 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 iseffective for DAR determination and monitoring of conjugation reactions.

1. Myoglobin
Competitor’sHIC column
2.5 µm
2. Ribonuclease
AmAU 3. Lysozyme
120
4.α-Chymotrypsinogen A

BioPro HIC BF 4 µmmAU
1201100804Rs (1,2)= 4.57
Rs (2,3)= 3.49
Rs (3,4)= 11.17

60
40

100

33.3-4.6 MPa

80

3Rs (1,2)= 2.78
4
Rs (2,3)= 4.37
160 Column:100
X
4.6mmI.D.
Rs (3,4)= 10.87
2Eluent: A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing
4022.0 M (NH4)2SO4B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0)0-100%B (0-11 min), 100%B (11-15 min)
0 L171212D02
Flow rate: 0.5 mL/min
0.0
2.5
5.0
7.5
10.0
12.5
15.0
Temperature : 25°CDetection: UV at 280 nmInjection: 15 μL

20200

7.3-10.5 MPa

L171208D02

2.5

0.0

5.0

7.5

10.0

12.5

15.0

17.5min

17.5

Competitor’s HIC column 2.5 µmmAU
120
1003

7.3-10.5 MPa

80

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

6040

1The separation of proteins is compared on BioPro HIC BF withparticle size 4 μm and a competitor’s HIC column with particle size
2.5 μm. BioPro HIC BF with optimized media design and packing
technology has superior resolution comparable to competitor’scolumn with sub-3 μm.

42200
17.5

min

L171212D02

0.0

2.5

5.0

7.5

10.0

12.5

15.0

17.5

min

Excellent peak shape under high loadingminHydrophobic Interaction Chromatography Column0High resolution comparable to sub-3 µm with low operating pressure

ColumnEluent: BioPro HIC BF 4 µm, 100 X 4.6 mmI.D.
: A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0)containing 2.0 M (NH4)2SO4B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0)
60%B (0-0.5 min), 60-100%B (0.5-7.5 min),
100%B (7.5-10 min)Flow rate: 1.2 mL/min
Temperature : 30℃Detection: UV at 280 nm
Sample: Humanized monoclonal IgG (2.5 mg/mL)

mAULoading— 80 μg— 200 μg
— 400 μg
— 800 μg

1250

1000

750

500

250

L180214A03
L180214A02
L180214A01
L180213C01

0
0.0

1.0

2.0

3.0

4.0

5.0

6.0

7.0

8.0

9.0minBioPro HIC BF provides superior peak shapes, even underhigh loading conditions. This allows detection of very tiny
amounts of impurities in the sample. In addition, it can beused for laboratory scale purifications, e.g. for isolation ofvariants for various research requirements (e.g. structuralanalysis).

Ordering Information –Column–
BioPro HIC BF
Particle size(µm)

Column size
inner diameter X length(mm)Product number44.6 X 100

BHB00S04-1046WT39