Hydrophobic Interaction Chromatography Column(HIC) 疏水性作用層析管柱
BioPro HIC HT以及BioPro HIC BF均為無孔疏水性聚合物(non-porous hydrophobic polymer)。 專門設計用來分析或實驗室級純化(lab-scale purification)抗體(antibodies)、抗體-藥物偶聯物(antibody-drug conjugates)和其他蛋白質(proteins)的樣品。
最佳化過的表面修飾和管柱填充方法可提高分離度(separation performance)。 使用BioPro HIC HT可以實現更快流速的高通量分析和高分辨率。 與其他HIC層析管柱相比，該硬質(rigid)顆粒具有更高的耐壓性，因此可以使用更高的流速，進而縮短分析時間。
| Matrix | Bonded phase |
Particle size (µm) |
Usable pH range |
Usable temperature range |
Characteristics | |
|---|---|---|---|---|---|---|
| BioPro HIC HT | Hydrophilic non-porous polymer |
Butyl group | 2.3 | 2-12 | 10-60°C | First-choice HIC column for high resolution/high throughput analyses |
| BioPro HIC BF | 4 | HIC column for separation of low hydrophobic proteins |
Comparison of retention behavior
BioPro HIC HT (first choice column) is suitable for successful separation of various biopharmaceuticals.
BioPro HIC BF shows the stronger retention of proteins than BioPro HIC HT due to the stationary phase hydrophobicity, and could be preferred for the separation of low hydrophobic proteins.
| Column | 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 0%B (0-1 min), 0-100%B (1-11 min), 100%B (11-15 min) |
| Flow rate | 0.5 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 15 µL |
| Sample | 1. Adalimumab (0.5 mg/mL) 2. Trastuzumab (0.5 mg/mL) |
BioPro HIC HT
-Suitable for analysis of biopharmaceuticals such as antibody-drug conjugates (ADC’s)
-High throughput by shortening analysis time
-Innovative surface chemistry leading to ideal drug-to-antibody ratio (DAR) analysis
-Excellent batch-to-batch reproducibility
-High throughput by shortening analysis time under high flow rate condition
BioPro HIC HT consists of the fine non-porous polymer particle designed for separation of proteins, and has high pressure tolerance with excellent chromatographic resolution by optimized packing condition. This is capable of the rapid analysis without losing the resolution at a high pressure, which is too high to use with the other commercially available HIC column.
| Eluent | A) 20 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 1.0 M (NH4)2SO4 B) 20 mM NaH2PO4-Na2HPO4 (pH 7.0)/2-propanol (85/15) |
|---|---|
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 10 µL |
| Sample | Brentuximab vedotin (2.5 mg/mL) |
-Innovative surface chemistry for drug-to-antibody ratio (DAR) analysis
The DAR analysis by using BioPro HIC HT and the competitor columns under two mobile phase conditions varying 2-propanol content was carried out. The competitor A column having high hydrophobicity did not elute the peak #5 completely with the mobile phase containing 2-propanol lower than 15%. The competitor B column eluted all peaks with the 10% 2-propanol mobile phase condition, but it showed the poor separation between the peaks #4 and #5. On the other hand, BioPro HIC HT eluted all peaks completely with the good separation, and another peak was partially separated on the peak #3. Additionally, it is noteworthy that the same DAR values were observed at any content of 2-propanol.
| Column | 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 20 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 1.0 M (NH4)2SO4 B) 20 mM NaH2PO4-Na2HPO4 (pH 7.0)/2-propanol (90/10) or (85/15) 0-100%B (0-15 min), 100%B (15-20 min), 0%B (20-35 min) |
| Flow rate | 0.5 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 10 µL |
| Sample | Brentuximab vedotin (2.5 mg/mL) |
-Excellent batch-to-batch reproducibility
BioPro HIC HT exhibits an excellent batch-to-batch reproducibility. This is suitable for quality control analysis of biopharmaceuticals such as MAb’s.
| Column | BioPro HIC HT 2.3 μm, 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 0%B (0-1 min), 0-100%B (1-11 min), 100%B (11-15 min) |
| Flow rate | 0.5 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 15 µL |
| Sample | 1. Adalimumab (0.5 mg/mL) 2. Trastuzumab (0.5 mg/mL) 3. Bevacizumab (0.5 mg/mL) |
BioPro HIC BF
-Higher hydrophobicity of HIC stationary phase
BioPro HIC BF shows the stronger retention of proteins due to the higher hydrophobicity of its stationary phase compared to the other commercially available HIC columns. This indicates that BioPro HIC BF could be preferred for the separation of low hydrophobic proteins.
| Column | 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 0%B (0-1 min), 0-100%B (1-11 min), 100%B (11-15 min) |
| Flow rate | 0.5 mL/min |
| Temperature | 25ºC |
| Detection | UV at 280 nm |
| Injection | 15 µL |
| Sample | 1. Adalimumab (0.5 mg/mL) 2. Trastuzumab (0.5 mg/mL) |
-Designed for separation of low hydrophobic proteins
Analysis of oxidized MAb samples, an example for biopharmaceutical characterization, is shown here.
BioPro HIC BF having strong hydrophobic retention could be preferred for such analysis because the oxidized MAb variants elute earlier than the nonoxidized MAb.
Analysis of oxidized MAb
NISTmAb was treated with tert-butyl hydroperoxide (t-BHP) as an oxidant in order to promote the oxidation. The oxidized MAb was analyzed by using BioPro HIC BF column.
Under the ammonium sulfate condition (a), four peaks appeared at earlier elution times compared to the peak of the nonoxidized MAb, presumably due to the conformational changes via the oxidization of the methionine residues.
Under the sodium chloride condition (b), eight peaks appeared at earlier elution times compared to the peak of the nonoxidized MAb. Such a better resolution was achieved with the shorter analysis time compared with under the ammonium sulfate condition (a).
| Column | BioPro HIC BF 4 µm, 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing salt B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 40-80%B (0-40 min), 80%B (40-45 min) |
| Flow rate | 0.3 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 5 µL (1.0 mg/mL) |
Analysis of papain-digested oxidized MAb
The papain-digests of NISTmAb samples with and without the oxidization were analyzed by using BioPro HIC BF column.
The Fab and Fc fragments were characterized from the chromatogram of the papain-digested MAb.
In the chromatogram of the papain-digested oxidized MAb, the multiple peaks appeared at earlier elution times compared to the peaks assigned to the Fab and Fc fragments, and would be corresponding to the oxidized fragments.
According to the previous report*, the oxidized fragments elute earlier than the nonoxidized ones.
*Journal of Chromatography A, 2008, 1214, 81-89
| Column | BioPro HIC BF 4 µm, 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) 40-80%B (0-10 min) |
| Flow rate | 1.0 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 5 µL (0.5 mg/mL) |
-Excellent peak shape under high loading condition
| Column | BioPro HIC BF 4 µm, 100 X 4.6 mmI.D. |
|---|---|
| Eluent | A) 100 mM NaH2PO4-Na2HPO4 (pH 7.0) containing 2.0 M (NH4)2SO4 B) 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°C |
| Detection | UV at 280 nm |
| Sample | Humanized monoclonal IgG (2.5 mg/mL) |
BioPro HIC BF shows excellent peak shape even under high loading conditions. This leads to effective for laboratory-scale purification and detection of minor constituents by the large volume injection.
Care and Use Manual
Product Information
-BioPro_HIC_HT
-BioPro_HIC_BF
-YMC Biochromatography Columns
-BioPro HIC HT by YMC for High Throughput
-Analytical Stationary Phases for BioLC from YMC-Overview
-The Principles of BioLC & YMC BioLC Phase Overview
-Characterisation of MAbs and related products via HIC-MS
-YMC Alternatives to Existing BioLC Columns
-YMC’s HIC Phase Especially Designed for Antibodies, ADCs and Proteins(請聯繫我們索取資料)
Technical Note
Application Note
-Analysis of oxidised monoclonal antibodies by hydrophobic interaction chromatography(U190422AE)
-HIC Analysis of Different Monoclonal Antibodies using Isopropanol as Modifier
-HPLC Analysis of Drug-to-Antibody Ratio with BioPro HIC BF by YMC
-High throughput DAR determination of brentuximab vedotin using YMC’s BioPro HIC HT
-Online native HIC-MS analysis of MAbs and their molecular variants using BioPro HIC BF
-Online native HIC-MS analysis of cys-linked ADCs using BioPro HIC BF
-The right HIC column for each separation- BioPro HIC HT and BioPro HIC -BF
| Packing material | Particle size
(µm) |
Pore size | Column size
inner diameter x length (mm) |
Product number |
|---|---|---|---|---|
| BioPro HIC HT | 2.3 | Non-porous | 4.6 X 100 | BHH00SQ3-1046PTH |
| BioPro HIC BF | 4 | Non-porous | 4.6 X 100 | BHB00S04-1046WT |
Please contact us about the product with other sizes.
