As an example of exosome (EV) analysis, the EV preparation process from cell culture supernatant was followed by a combination of polymer-based aqueous SEC (GFC) column OHpak SB-806 HQ and various detectors. UV 280 nm covers general culture-derived impurities, and fluorescence (Ex 280 nm / Em 348 nm) responds mainly to proteins via Trp residue fluorescence. In addition, MALS scattered light (LS) provides a highly sensitive response especially for large objects like nanoparticles. Also, MALS gives an estimate of the target RMS (root mean square) radius.
The fraction consisted mainly EV was separated from many culture-derived impurity components and was found around 8 minutes. While UV and fluorescence provide important insights into the progress and efficacy of the purification process and profiling of purified products, they are less sensitive to EVs mostly composed of lipid membranes and containing trace amounts of protein / nucleic acid cargo. It can be seen that LS is an effective EV tracking method, especially in the early stages of purification.
SB-806 HQ is a high-performance aqueous SEC (GFC) column suitable for bioproducts with a sufficient pore size to hold and separate EV-class nanoscale objects. Combined with a variety of detectors, it is useful for comprehensive analysis of the complex bio-nano target preparation process.
EV fraction preparation conditions
(1) Concentration step: Commercially available centrifugal ultrafiltration membrane 100 kDa (40 times concentrated)
(2) Affinity step: Commercial affinity purification kit (equivalent to 10-fold concentration)
Cooperation of sample preparation by Showa Denko Materials Co., Ltd.
Sample :
1. Cell culture supernatant 50 μL inj.
2. Concentrate 50 μL inj.
3. Crude product 15 μL inj.
Column : Shodex OHpak SB-806 HQ (8.0 mm I.D. x 300 mm) Eluent : PBS (-) Flow rate : 1.0 mL/min Detector : UV (280 nm) Fluorescence (Ex.280 nm, Em.348 nm) MALS (DAWN8+ produced by Wyatt Technology Corp.) Column temp. : 25 °C
Sample Name Index
Operation Manual / Certificate of Analysis
Operation Manuals and Certificate of Analysis / Inspection Certificate for the following products can be downloaded here.
Product Name Index
Applications
- Sample Load (KW-803)
- Effects of Flow Rate (KW-802.5, KW-803)
- Standard Proteins (1) (KW-802.5)
- Standard Proteins (2) (KW-802.5 and KW-803)
- Standard Proteins (14) (KW-802.5)
- Standard Proteins (15) (KW-803)
- Standard Proteins (16) (KW-804)
- Standard Proteins (18) (KW-803)
- Standard Proteins (19) (KW-804)
- Standard Proteins (23) (GF-510 HQ)
- Standard Proteins (24) (KW402.5-4F)
- Standard Proteins (25) (KW403-4F)
- Standard Proteins (26) (KW404-4F)
- Standard Proteins (27) (KW405-4F)
- Recovery of Proteins (GF-310 HQ and GS-320 HQ)
- Recovery of Proteins (KW-802.5 and KW-803)
- Recovery of Proteins (KW-803)
- Proteins in Human Serum (3) (KW-803)
- Dimer and Trimer of BSA
- Crude Albumin (Chicken Egg) (1) (KW-803)
- Crude Lipoxidase
- Crude Catalase
- Crude Hexokinase
- Human Serum (1) (SB-802 HQ)
- Tryptic Digest of Human IgG
- Comparison of Chromatograms of Protein mixtures
- Comparison between KW402.5-4F and KW-802.5
- Recovery of Proteins (KW402.5-4F, KW403-4F)
- Human Control Serum
- Analysis of Whey in Yogurt (KW403 4F + KW402.5 4F)
- Lectin Protein
- Peptide Analysis (SEC)
- PEG-Chymotrypsin (KW-802.5)
- SEC Analysis of PEG-Asparaginase (SB-806 HQ)
- Human IgM (2)
- Poly-Lysine
- Avidin (2)
- α1 Acid Glycoprotein
- Analysis of Gelatin (SB-806M HQ)
- Analysis of Insulin Glargine According to JP Method (KW-802.5)
- Analysis of Human Insulin According to JP Method (KW-802.5)
- Analysis of High Molecular Impurities in Insulin Aspart According to Pharmacopeia Method (KW-802.5)
- Analysis of High Molecular Weight Protein According to USP-NF GENERAL CHAPTERS Physicochemical Analytical Procedures for Insulins (KW-802.5)
- Comparison of SEC Separation of Standard Proteins
- Comparison of SEC Separation of Polyclonal Antibody
- Comparison of SEC Separation of Monoclonal Antibody
- Comparison of Separation of BSA Aggregates (LW-803, KW-803)
- Monitoring Papain Digestion of Humanized Monoclonal IgG (LW-803)
- Analysis of Lipoproteins in Serum (KW-804)
- Comparison of SEC Separation of Standard Proteins between LW-403 4D and LW-803
- Durability Test by Number of Sample Injections (LW-403 4D)
- Analysis of Epoetin According to USP-NF Method (LW-803)
- Comparison of Separation of Proteins with Conventional Device and Semi-micro Type Device (LW-403 4D)
- Comparison of IgG Separation with Conventional Device and Semi-micro Type Device (LW-403 4D)
- Particle Size Distribution Determination of Hepatitis B Surface Antigen Virus-like Particle (HBsAg VLP) (KW405-4F)
- Analysis of Hemocyanin (KW405-4F)
- Analysis of Somatropin According to EP Method (LW-803)
- SEC Analysis of Rituximab (LW-803)
- SEC/MALS Analysis of Norovirus VLP (SB-805 HQ)
- Analysis of Thymosin α1 (KW-802.5)
- Analysis of Proteoglycan in a Supplement (SB-806M HQ)
- Molecular Weight Measurements of Low Molecular Weight Heparins According to USP-NF GENERAL CHAPTERS <209> (KW-803 + KW-802.5)
Applications (Related Information)
- Calibration Curves for KW-800 Series (Pullulan)
- Calibration Curves for KW-800 Series (Proteins)
- Calibration Curves for KW-800 Series (PEG, PEO)
- Calibration Curves of KW400-4F (1) (Pullulan)
- Calibration Curves of KW400-4F (2) (Proteins)
- Calibration Curves of KW400-4F (3) (PEG, PEO)
- Calibration Curves for GF-HQ Series (Aqueous Eluent: Proteins)
- Calibration Curve for LW-803 (Proteins)
- Calibration Curve for LW-403 4D (Proteins)