[PDF] Présentation PowerPoint Column : Bio SEC-5 1000Å

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1Size Exclusion

BioHPLC Columns

NEW Size Exclusion Columns

•5mm Particle •100 Å, 150 Å, 300 Å, 500 Å, 1000 Å,

2000 Å pore sizes

•High stability and long lifetime •Great reproducibility Agilent Bio SEC-5High Capacity and High Resolution Min12 21334
45

5Columns: Bio SEC-5, 4.6x300 mm

Buffer: 0.15 M Phosphate buffer, pH 7.0

Flow rate: 0.35 mL/min

Detector: UV 214 nm

Injection volume: 10 µL1.Thyroglobulin

2. g-Globulin

3. Ovalbumin

4. Ribonuclease A

5. p-Aminobenzoic acid

0.002.004.006.008.0010.0012.0014.001

234
5

Bio SEC-5, 150Å

Bio SEC-5, 300Å

Bio SEC-5, 500Å

Agilent Bio SEC-5High Molecular Weight Proteins

Proteins :

1.Thyroglobulin (670kD)

2. Uracil (120D)Column : Bio SEC-5, 1000Å, 4.6x300mm

Buffer : 150 mM phosphate buffer, pH 7.0

Flow rate : 0.35 mL/min

Detection : 214 nm

Temperature : Ambient

1 Possible DimerAU0.000.020.040.060.080.100.120.140.160.180.200.22

Min024681012142

Agilent Bio SEC-5Reproducibility and Lifetime

12

4Agilent Bio SEC-5, 300Å, 7.8x300mm

Buffer: 150 mM Phosphate buffer , pH 7.0

Flow Rate : 1.0 mL/min

Detector : 214 nm

Injection : 10 uL1.Thyroglobulin

2. BSA

3. Ribonuclease A

4. Uracil

Minutes3

Agilent Bio SEC-5

Batch (Lot) ReproducibilityAU0.000.100.200.300.400.500.600.700.80

Min024681012141

23

43rd run

After 300

injections

Min024681012141

234
Lot 1 Lot 2

Lot 3150Å, 7.8x300mm

150 mM Phosphate

Buffer, pH 7.0

1.Thyroglobulin

2. BSA

3. Ribonuclease A

4. Uracil

Agilent Bio SEC-5Lot to Lot Reproducibility, 300Å, 7.8x300mm

LotThyroglobulinBSARibonuclease AUracil

RTEff.RTEff.RTEff.RTEff.

102506CL6.7716039.29651510.471181212.2126709Black : SN 14086

Lot# 050907MGB

Blue : SN 14045

Lot# 102606MG

Green : SN 13247

Lot# 102506CLAU0.000.020.040.060.080.100.120.140.160.180.20

Agilent Bio SEC-5Stability at pH 8.5

Pre-test, pH7.0

Day 1 AM, pH8.5

Day 1 PM, pH8.5

Day 2 AM, pH8.5

Day 2 PM, pH8.5

Day 3 AM, pH8.5Bio SEC-5, 300Å, 4.6x300mm

Buffer : 150mM Phosphate, pH 8.5

Wavelength : 214 nm

Flow Rate : 0.35 mL/min

Injection : 3 uL

Minutes0123456789101112131415Day 7 AM, pH8.51. Thyroglobulin

2. BSA

3. Ribonuclease A

4. Uracil12

34
Agilent Bio SEC-5Monoclonal Antibody Aggregation Monitoring

Columns : Bio SEC-5, 300Å, 7.8x300mm

Buffer : 150 mM PBS, pH 7

Flow Rate : 1.0 mL/min

Sample : Mab (2.5 mg/mL)

Injection : 10 mL

Concentration : 10, 2.5, 0.5 mg/mL

(from high to low) Temperature : AmbientAU0.000.200.400.600.801.001.201.401.601.80 Agilent Bio SEC-5Monoclonal Antibody Aggregation Monitoring

Columns : Bio SEC-5, 300Å, 7.8x300mm

Buffer : 150 mM PBS, pH 7

Flow Rate : 1.0 mL/min

Sample : Mab (2.5 mg/mL)

Injection : 10 mL

Concentration : 10, 2.5, 0.5 mg/mL

(from high to low)

Temperature : AmbientAU

-0.04 -0.02 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14

Minutes

Agilent Bio SEC-5Monoclonal Antibody Aggregation Monitoring

MAb Dimer

Mab AggregatesColumns : Bio SEC-5, 300Å, 7.8x300mm

Buffer : 150 mM PBS, pH 7

Flow Rate : 1.0 mL/min

Sample : Mab

Temperature : AmbientAU-0.008-0.006-0.004-0.0020.0000.0020.0040.0060.0080.0100.0120.0140.0160.0180.020

Min1.02.003.04.05.06.07.08.09.010.011.012.013.014.015.0Buffer Peak

Low molecular

weight forms Min0123456789101112131415Columns : Bio SEC-5, 7.8x300mm

Buffer : 150 mM Phosphate, pH 7.0

Flow Rate : 1.0 mL/min

Detector : 214 nm

Injection : 10 µL (2.5 mg/mL)

SEC-5, 1000Å

SEC-5, 500Å

SEC-5, 300Å

SEC-5, 150ÅAgilent Bio SEC-5Size Exclusion Separations of E. coli Lysate Columns : Bio SEC-5, 1000Å, 7.8x300mm + Bio SEC-5, 150Å, 7.8x300mm

Buffer : 150 mM Phosphate, pH 7.0

Flow Rate : 1.0 mL/min

Detector : 214 nm

Injection : 10 µL (2.5 mg/mL)AU0.00.20.40.60.81.01.21.4 Minutes024681012141618202224262830Agilent Bio SEC-5Size Exclusion Separations of E. coli Lysate (Two Columns In Series)

Agilent Bio SEC-5Column Characteristics

Agilent Bio SEC-5Ordering Information

NEW Size Exclusion Columns

•Unique, 3 mm particle •100 Å, 150 Å, 300 Å pore sizes •Highest resolution •Highest efficiency •Faster SEC separations

Agilent Bio SEC-3

Improved Efficiency With Smaller

Particles

Column : Bio SEC-3 300Å and Bio SEC-5 300Å

Buffer : 150 mM Phosphate buffer, pH 7

Flow rate : 1.0 mL/min for 7.8x300 mm

Temperature : Ambient (~23° C)

Detection : UV 214nm

Injection : 10 µL (3 mL for 4.6x300 mm)

Sample : 1) Thyroglobulin (1.0 mg/mL), 670 kD;

2) BSA dimer, 132 kD; 3) BSA (1.0 mg/mL), 66 kD;

4) Ribonuclease A (1.0 mg/mL), 13.7 kD, and 5) Uracil

(2.5 mg/mL), 120D.Min0123456789101112131415Agilent Bio SEC-3, 300Å,

7.8x300mm

Agilent Bio SEC-3, 300Å,

7.8x300mmColumnPressure

Bio SEC-3

(7.8x300mm)1350 psi

Bio SEC-5

(7.8x300mm)650 psiPeakProteinSEC-3, 300Å (7.8x300mm)SEC-5, 300Å (7.8x300mm)

1Thyroglobulin24601120

2BSA Dimer51002720

3BSA 130906590

4Ribonuclease A2200011160

5Uracil3850027860

Agilent Bio SEC-3Sharp Peaks and High Resolution of Proteins

Columns : Bio SEC-3, 7.8x300 mm

Mobile phase : 150 mM Phosphate, pH 7

Flow rate : 1.0 mL/min

Temperature : Ambient (~23° C)

Detection : UV 214nm

Injection : 10 µL

Sample :

1)Thyroglobulin (1.0 mg/mL), 670 kD

2)BSA dimer, 132 kD

3)BSA (1.0 mg/mL), 66 kD

4)Ribonuclease A (1.0 mg/mL), 13.7 kD

5)Uracil (2.5 mg/mL), 120DPeakCompoundSEC-3 150ÅSEC-3 300Å

1Thyroglobulin180502460

2BSA Dimer24205110

3BSA562013090

4Ribonuclease A1760022000

234
1

234Agilent Bio SEC-3, 150Å,

7.8x300mm

Agilent Bio SEC-3, 300Å,

7.8x300mm5

5 Agilent Bio SEC-3Separation of Bio-Rad Size Exclusion Protein Standard

Columns : Bio SEC-3, 150Å, 7.8x300mm

& Bio SEC-3 300Å, 7.8x300mm

Buffer : 0.15 M Phosphate, pH 7.0

Flow rate : 1.0 mL/min

Detector : 214 nm

Injection : 10 µL

Sample :

1)Thyroglobulin, 670 kD

2)g-Globulin, 158 kD

3)Ovalbumin, 44 kD

4)Myoglobulin, 16.9 kD

5)Vitamin B12, 1355 D. Peak

ProteinSEC-3

150ÅSEC-3

300Å

1Thyroglobulin124201760

2g-Globulin28603650

3Ovalbumin662011760

4Myoglobulin1502020810

5Vitamin B123437035460Min01234567891011121314151

234
5 1

2345Agilent Bio SEC-3, 150Å,

7.8x300mm

Agilent Bio SEC-3, 300Å,

7.8x300mm

Agilent Bio SEC-3Separation of Protein and Peptide Mixture

Min01234567891011121314151

234
56
1 234

56Agilent Bio SEC-3,

150Å, 7.8x300mm

Agilent Bio SEC-3,

300Å, 7.8x300mmColumn : Bio SEC-3 150Å, 7.8x300mm &

Bio SEC-3 300Å, 7.8x300mm

Mobile phase : 150 mM Phosphate, pH 7 ;

Flow rate : 1.0 mL/min

Temperature : Ambient (~23° C); Detection:

UV 214nm

Sample :

1) Thyroglobulin, 670kD;

2) BSA monomer, 66kD;

3) Ribonuclease A, 13.7kD;

4) poly-DL-alanine, 1-5 kD;

5) Uracil, 120D.

Injection : 10 µL

Agilent Bio SEC-3Bio SEC-3, 150Å, Capacity: BSA Loading TestAU0.000.200.400.600.801.001.201.401.601.802.00

Min0.01.02.03.04.05.06.07.08.09.010.011.012.013.014.015.0Injection volume : 10 mL

BSA Concentration

(from low to high) :

1 mg/mL

2 mg/mL

5 mg/mL

10 mg/mL

20 mg/mL

50 mg/mLColumn : Bio SEC-3, 150Å, 7.8x300mm

Mobile phase : 150 mM Phosphate, pH 7.0

Flow Rate : 1.0 mL/min

Injection volume : 10 µL

Detector : 214 nm

Agilent Bio SEC-3Monoclonal Antibody Aggregation Monitoring

Column : Bio SEC-3 300Å, 7.8x300mm

Mobile phase : 150 mM Phosphate, pH 7

Flow rate : 1.0 mL/min

Temperature : Ambient

Sample : Monoclonal antibody (10 mL, 5 mg/mL)AU0.000.050.100.150.200.250.300.350.400.450.500.55 Agilent Bio SEC-3Monoclonal Antibody Aggregation MonitoringAU -0.005 0.000 0.005 0.010 0.015 0.020 0.025 0.030 0.035

Minutes

1.002.003.004.005.006.007.008.009.0010.0011.0012.0013.0014.0015.00Column : Bio SEC-3 300Å, 7.8x300mm

Mobile phase : 150 mM Phosphate, pH 7

Flow rate : 1.0 mL/min

Temperature : Ambient

Sample : Monoclonal antibody (10 mL, 5 mg/mL)

Possible

AggregatesDimerMonomer

Agilent Bio SEC-3Separations of E. coli lysate

Column : Column: Bio SEC-3 150Å, 7.8x300mm & Bio SEC-3 300Å, 7.8x300mm

Mobile phase : 0.15 M Phosphate, pH 7.0

Flow rate : 1.0 mL/min

Detection : UV 214 nm

Temperature : Ambient

Injection : 10 µL

Sample : E. coli lysate (2.5 mg/mL) Min0.01.02.03.04.05.06.07.08.09.010.011.012.013.014.015.0Bio SEC-3 150Å, 7.8x300mm

Bio SEC-3 300Å, 7.8x300mm

Agilent Bio SEC-3Column Characteristics

Agilent Bio SEC-3Ordering Information

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