YMC-SEC MAB/YMC-Pack Diol 分子篩層析管柱(Size exclusion chromatography, SEC) /凝膠過濾層析管住(Gel filtration chromatography, GFC)
YMC提供適合分離分子量200至數十萬的各種胜肽(peptides)，蛋白質(proteins)，寡核苷酸(oligonucleotides)，碳水化合物(carbohydrates)和其他生物聚合物(biopolymers)的分子篩層析管柱。
YMC-Pack Diol 使用1,2-dihydroxypropane於silica固定相上進行修飾，並設計各種可用之孔徑，藉由嚴格控制的孔隙度(porosities)可進行大範圍的胜肽(peptide)到大分子蛋白質(proteins)或抗體(antibodies)的分析。優點包括平衡時間短(shorter equilibration time)、良好選擇性(good selectivity)、可耐高壓、價格實惠、可將HPLC方法轉移至UHPLC以及二次作用力低(secondary interactions)。
YMC-SEC MAB專為分析抗體單體(antibodies monomers)及其聚集體/片段(aggregates/fragments)和聚集體(aggregates)而設計，可為高分子量和低分子量範圍提供高分辨率(high resolution)、穩定性高(excellent durability)、批次間重複性佳(batch-to-batch reproducibility)。其最佳化過之表面修飾，大幅降低該類樣品之非特異性吸附(nonspecific adsorption)。是針對抗體藥物品質管控之理想選擇。
規格如下：
Specification
| YMC-SEC MAB | YMC-Pack Diol-60 |
YMC-Pack Diol-120 |
YMC-Pack Diol-200 |
YMC-Pack Diol-300 |
|
|---|---|---|---|---|---|
| Base | Silica gel | ||||
| Bonded phase | Dihydroxypropyl (Diol) | ||||
| Particle size (µm) | 3 | 3, 5 | 2, 3, 5 | ||
| Pore size (Å) | 250 | 60 | 120 | 200 | 300 |
| Usable pH range | 5-7.5 | ||||
| Molecular weight range | 10,000-700,000 |
Below 10,000 |
1,000-100,000 |
5,000-300,000 |
20,000-1,000,000 |
分離原理
Size Exclusion Chromatography (SEC), also referred to as Gel Filtration Chromatography (GFC) or Gel Permeation Chromatography (GPC), is a mode of liquid chromatography in which the components of a mixture are separated according to differences in their size. It is an important tool for the analysis and separation of natural and synthetic biopolymers.
Molecules with shapes such as rigid rods, random chains and spheres but with the same molecular weight behave differently in SEC. The principle of separation is based on differences in the hydrodynamic radius of the molecules in solution.
Molecules with a larger radius elute earlier and those with the smallest radius are retained longer. The separation limit is such that only those compounds which differ by more than 10% in MW can be separated by SEC.
Large molecules exit the column more rapidly as they cannot permeate the porous structure of stationary phase. Smaller ones with the lowest hydrodynamic volume elute with longer retention times because they are able to penetrate some or all of the pores of the stationary phase. Molecules of intermediate size elute in an intermediate position.
-Suitable for separation of aggregates of Antibodies or Antibody Drug Conjugates (ADCs).
A humanized monoclonal antibody and an ADC were analyzed using YMC-SEC MAB. Chromatograms show excellent peak shape, and good separation between aggregates and a monomer peak. YMC-SEC MAB is effective for characterization and quality control of antibody drugs.
Analysis of a humanized monoclonal antibody
| Column | 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 | 25°C |
| Detection | UV at 280 nm |
| Injection | 10 µL |
| Sample | Humanized monoclonal antibody (5 mg/mL) |
Analysis of an antibody-drug conjugate (ADC)
| Column | 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/2-propanol (85/15) |
| Flow rate | 0.165 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 4 µL |
| Sample | Cystein-conjugated ADC mimic (2.5 mg/mL) |
-Excellent durability
YMC-SEC MAB has excellent repeatability of separation without any change of theoretical plate number and elution time even after repeated separation of a monoclonal antibody for more than 500 times because of its superior durability.
| Column | YMC-SEC MAB (3 µm, 250 Å) 300 X 4.6 mmI.D. |
|---|---|
| Eluent | 0.1 M NaH2PO4-Na2HPO4 (pH 7.0) containing 0.2 M NaCl |
| Flow rate | 0.33 mL/min |
| Temperature | 25°C |
| Detection | UV at 280 nm |
| Injection | 2 µL |
| Sample | Humanized monoclonal antibody (1 mg/mL) |
-Superior batch-to-batch reproducibility
YMC-SEC MAB provides excellent reproducibility of the separation of monomer and aggregates as well as for monomer and fragments. Therefore, it is very effective for quality control of antibody drugs.
| Column | 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 | 25°C |
| Detection | UV at 280 nm |
| Injection | 10 µL |
| Sample | Humanized monoclonal antibody (5 mg/mL) |
YMC-Pack Diol
-Calibration curves of various proteins
Comparison with different pore sizes
Diol-120, Diol-200, and Diol-300 are suitable for the separation or molecular weight determination of proteins with molecular weights of 10,000 to several hundred thousands.
| Column | YMC-Pack Diol 5 µm, 300 X 8.0 mmI.D. |
|---|---|
| Eluent | 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 |
Comparison with different particle sizes
YMC-Pack Diol column has an identical calibration curve across all of the particle sizes including 2 µm. This would realize smooth method transfer between HPLC and UHPLC.
| Column | YMC-Pack Diol, 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 | 25°C |
| Detection | UV at 280 nm |
-Analysis of plasma contituents
| Eluent | 0.1M KH2PO4-K2HPO4 (pH 7.0) containing 0.2M NaCl |
|---|---|
| Flow rate | 0.5 mL/min |
| Temperature | ambient (25°C) |
| Detection | UV at 280 nm |
Excellent durability
YMC-Pack Diol column maintained its performance for more than 500 injections on monoclonal antibody analysis.
| Column | YMC-Pack Diol-300 2 µm 150 X 4.6 mmI.D. |
|---|---|
| Eluent | 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 |
-For separation of peptides with molecular weights of 10,000 or less
| Column | YMC-Pack Diol-60 5 µm, 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 |
-For molecular weight determination of sugars
| Column | YMC-Pack Diol 5 µm, 500 X 8.0 mmI.D. |
|---|---|
| Eluent | water |
| Flow rate | 1.0 mL/min |
| Temperature | ambient |
| Detection | RI |
-Excellent reproducibility
YMC-Pack Diol column has excellent lot-to-lot reproducibility. This feature is ideal for quality control of protein-based drugs including monoclonal antibodies.
1. Thyroglobulin
2. γ-Globulin
3. Ovalbumin
4. Myoglobin
5. Vitamin B12
| Column | YMC-Pack Diol-300 5 µm, 300 X 8.0 mmI.D. |
|---|---|
| Eluent | 0.1 M NaH2PO4-Na2HPO4 (pH 7.0) containing 0.2 M NaCl |
| Flow rate | 1.0 mL/min |
| Temperature | ambient |
| Detection | UV at 280 nm |
| Application | Columns | Compound name | |
| Casein | YMC-Pack Diol (SEC) | Casein | |
| Chondroitin sulfate A | YMC-Pack Diol (SEC) | Chondroitin sulfate A, Na salt | |
| Chondroitin sulfate B | YMC-Pack Diol (SEC) | Chondroitin sulfate B, Na salt | |
| Chondroitin sulfate C | YMC-Pack Diol (SEC) | Chondroitin sulfate C, Na salt | |
| Chondroitin | YMC-Pack Diol (SEC) | Chondroitin, Na salt | |
| Cyclodextrins | YMC-Pack Diol (SEC) | γ-Cyclodextrin β-Cyclodextrin α-Cyclodextrin | |
| Cytochrome c | YMC-Pack Diol (SEC) | Cytochrome c (hexamer) Cytochrome c (tetramer) Cytochrome c (trimer) Cytochrome c (dimer) Cytochrome c (monomer) | |
| Glycine oligomers | YMC-Pack Diol (SEC) | Pentaglycine Triglycine Glycine | |
| Heparan sulfate | YMC-Pack Diol (SEC) | Heparan sulfate, Na salt | |
| Heparin | YMC-Pack Diol (SEC) | Heparin, Na salt | |
| Human IgG (λ), myeloma protein | YMC-Pack Diol (SEC) | Human IgG (λ), myeloma protein | |
| Human IgM (κ), myeloma protein | YMC-Pack Diol (SEC) | Normal human IgM (κ), myeloma protein | |
| Human immunoglobulins | YMC-Pack Diol (SEC) | Human IgM (κ), myeloma protein Normal human IgA Normal human IgG | |
| Hyaluronic acid | YMC-Pack Diol (SEC) | Hyaluronic acid, Na salt | |
| Keratan polysulfate | YMC-Pack Diol (SEC) | Keratan polysulfate, Na salt | |
| Keratan sulfate | YMC-Pack Diol (SEC) | Keratan sulfate, Na salt | |
| Maltooligosaccharides (G1 ～ G15) | YMC-Pack Diol (SEC) | Maltopentadecaose (G15) Maltotridecaose (G13) Maltoundecaose (G11) Maltoheptaose (G7) Maltopentaose (G5) Maltotriose (G3) Glucose (G1) | |
| Maltooligosaccharides (G1 ～ G15) | YMC-Pack Diol (SEC) | Maltopentadecaose (G15) Maltotridecaose (G13) Maltoundecaose (G11) Maltoheptaose (G7) Maltopentaose (G5) Maltotriose (G3) Glucose (G1) | |
| Monoclonal antibody (MAb) | YMC-Pack Diol (SEC) | Mouse monoclonal IgG1 (anti-human IgG4) | |
| Nonionic surfactants | YMC-Pack Diol (SEC) | Triton X-100 | |
| Normal human IgA | YMC-Pack Diol (SEC) | Normal human IgA | |
| Normal human IgG | YMC-Pack Diol (SEC) | Normal human IgG | |
| Peptide fragments from myoglobin | YMC-Pack Diol (SEC) | myoglobin fragments 1-153 (MW 17000) 1-131 (MW 14500) 56-153 (MW 10700) 56-131 (MW 8200) 1-55 (MW 6300) 132-153 (MW 2500) | |
| Peptide mapping | YMC-Pack Diol (SEC) | Peptide mapping | |
| Peptides | YMC-Pack Diol (SEC) | Insulin (Bovine) Neurotensin Angiotensin II Glycine | |
| Plasma constituents | YMC-Pack Diol (SEC) | Human serumIgM IgA Fibrinogen γ-Globulin IgG Transferrin Albumin α1-Antitrypsin Carbonic anhydrase Lysozyme Uric acid Creatinine | |
| Plasmid pBR322 and pBR322 Hae III restriction fragments | YMC-Pack Diol (SEC) | Plasmid pBR322 (4361 bp) Plasmid pBR322 Hae III digest (8-587 bp) | |
| Plasmid pBR322 restriction fragments | YMC-Pack Diol (SEC) | Plasmid pBR322 cleaved with restriction endonuclease Msp I | |
| Polydextrose | YMC-Pack Diol (SEC) | Polydextrose II | |
| Polyethyleneglycols | YMC-Pack Diol (SEC) | PEG #20000 (MW 20000) PEG #6000 (MW 8400) PEG #4000 (MW 3350) PEG #1000 (MW 1000) PEG #300 (MW 300) Ethyleneglycol (MW 62) | |
| Proteins for molecular weight marker | YMC-Pack Diol (SEC) | Glutamate dehydrogenase (MW 290000) Lactate dehydrogenase (MW 142000) Enolase (MW 67000) Adenylate kinase (MW 32000) Cytochrome c (MW 12400) | |
| Proteins in human serum | YMC-Pack Diol (SEC) | IgG Transferrin Albumin | |
| Proteins in mouse ascites fluid | YMC-Pack Diol (SEC) | IgG Transferrin Albumin | |
| Pullulans | YMC-Pack Diol (SEC) | Pullulan P-800 (MW 853000) Pullulan P-50 (MW 48000) Pullulan P-20 (MW 23700) Pullulan P-5 (MW 5800) | |
| lgG, Fab and Fc fragments | YMC-Pack Diol (SEC) | Rabbit lgG Mouse lgG Fc fragment Mouse lgG Fab fragment |
Care and Use Manual
Product Information
Technical Note
Application Note
YMC-SEC MAB
| Packing material | Particle size
(µm) |
Column size
inner diameter x length (mm) |
Product number |
|---|---|---|---|
| YMC-SEC MAB | 3 | 4.6 X 300 | DLM25S03-3046WT |
| 8.0 X 300 | DLM25S03-3008WT |
Please contact us about the product with other sizes.
UHPLC Columns (Max. pressure : 45 MPa)
| Packing material | Particle size
(µm) |
Pore size
(Å) |
Column size
Inner diameter x length (mm) |
Product Number |
|---|---|---|---|---|
| Diol-200 | 2 | 200 | 4.6 X 150 | DL20S02-1546PTH |
| 4.6 X 300 | DL20S02-3046PTH | |||
| Diol-300 | 2 | 300 | 4.6 X 150 | DL30S02-1546PTH |
| 4.6 X 300 | DL30S02-3046PTH |
Analysis Columns (Stainless Columns, Max. pressure : 20 MPa)
| Packing material | Particle size
(µm) |
Pore size
(Å) |
Column size
Inner diameter x length (mm) |
Product Number |
|---|---|---|---|---|
| Diol-60 | 3 | 60 | 4.6 X 300 | DL06S03-3046WT |
| 5 | 60 | 4.6 X 300 | DL06S05-3046WT | |
| 8.0 X 300 | DL06S05-3008WT | |||
| 8.0 X 500 | DL06S05-5008WT | |||
| Diol-120 | 3 | 120 | 4.6 X 300 | DL12S03-3046WT |
| 5 | 120 | 4.6 X 300 | DL12S05-3046WT | |
| 8.0 X 300 | DL12S05-3008WT | |||
| 8.0 X 500 | DL12S05-5008WT | |||
| Diol-200 | 3 | 200 | 4.6 X 300 | DL20S03-3046WT |
| 5 | 200 | 4.6 X 300 | DL20S05-3046WT | |
| 8.0 X 300 | DL20S05-3008WT | |||
| 8.0 X 500 | DL20S05-5008WT | |||
| Diol-300 | 3 | 300 | 4.6 X 300 | DL30S03-3046WT |
| 5 | 300 | 4.6 X 300 | DL30S05-3046WT | |
| 8.0 X 300 | DL30S05-3008WT | |||
| 8.0 X 500 | DL30S05-5008WT |
Guard Columns (Stainless Columns)
| Packing material | Particle size
(µm) |
Pore size
(Å) |
Column size
Inner diameter x length (mm) |
Product Number |
|---|---|---|---|---|
| Diol-60 | 5 | 60 | 8.0 X 30 | DL06S05-0308WTG |
| Diol-120 | 5 | 120 | 8.0 X 30 | DL12S05-0308WTG |
| Diol-200 | 5 | 200 | 8.0 X 30 | DL20S05-0308WTG |
| Diol-300 | 5 | 300 | 8.0 X 30 | DL30S05-0308WTG |
Analysis Columns (Glass Columns, Max. pressure : 5 MPa)
| Packing material | Particle size
(µm) |
Pore size
(Å) |
Column size
Inner diameter x length (mm) |
Product Number |
|---|---|---|---|---|
| Diol-60 | 5 | 60 | 8.0 X 300 | DL06S05-3008FG |
| 8.0 X 500 | DL06S05-5008FG | |||
| Diol-120 | 5 | 120 | 8.0 X 300 | DL12S05-3008FG |
| 8.0 X 500 | DL12S05-5008FG | |||
| Diol-200 | 5 | 200 | 8.0 X 300 | DL20S05-3008FG |
| 8.0 X 500 | DL20S05-5008FG | |||
| Diol-300 | 5 | 300 | 8.0 X 300 | DL30S05-3008FG |
| 8.0 X 500 | DL30S05-5008FG |
Semi-prep Columns (Stainless Columns, Max. pressure : 10 MPa)
| Packing material | Particle size
(µm) |
Pore size
(Å) |
Column size
Inner diameter x length (mm) |
Product Number |
|---|---|---|---|---|
| Diol-60 | 5 | 60 | 20 X 300 | DL06S05-3020WT |
| 20 X 500 | DL06S05-5020WT | |||
| Diol-120 | 5 | 120 | 20 X 300 | DL12S05-3020WT |
| 20 X 500 | DL12S05-5020WT | |||
| Diol-200 | 5 | 200 | 20 X 300 | DL20S05-3020WT |
| 20 X 500 | DL20S05-5020WT | |||
| Diol-300 | 5 | 300 | 20 X 300 | DL30S05-3020WT |
| 20 X 500 | DL30S05-5020WT |
Semi-prep Guard Columns (Stainless Columns)
| Packing material | Particle size
(µm) |
Pore size
(Å) |
Column size
Inner diameter x length (mm) |
Product Number |
|---|---|---|---|---|
| Diol-60 | 5 | 60 | 20 X 50 | DL06S05-0520WTG |
| Diol-120 | 5 | 120 | 20 X 50 | DL12S05-0520WTG |
| Diol-200 | 5 | 200 | 20 X 50 | DL20S05-0520WTG |
| Diol-300 | 5 | 300 | 20 X 50 | DL30S05-0520WTG |
