Download Documents | |
SDS | USER MANUAL |
CellMosaic has designed this kit for antibody (IgG) labeling of SN38, the active metabolite of irinotecan, for early stage screening of your antibody.
Irinotecan is on the World Health Organization's List of Essential Medicines, the most effective and safe medicines needed in a health system. SN38 is 1000-times more active than irinotecan, but is not generally used as a drug due to its poor water solubility. If it is conjugated to a protein or antibody, the protein/antibody can transport SN38 to the active site. This kit includes O-succinyl SN38 NHS ester, which can be coupled to the antibody directly via surface amines in a single step. The product is then purified to remove any unreacted drug.
This kit provides materials to conjugate 1 to 3 mg of one (CM11408) or three (CM11408x3) antibody samples (IgG) with SN38.
If you have less than 1 mg of antibody, please add Antibody PerKit™ Customization for Lower Amount (AS1003) along with this kit.
If you would like CellMosaic to perform the conjugation instead, please add PerKit™ Custom Conjugation (AS1002) along with this kit.
Shipping policy: Ship at RT, choose overnight (ships Mon.-Thur.) or 2-day delivery (ships Mon.-Wed.) at checkout.
Drug Information
Requirement for Antibody
Key Features of this PerKit™
After conjugation: you can choose to send your sample to CellMosaic for HPLC analysis and calculation of the DAR.
Conjugation Chemistry
Protocol
Example of SN38 Labeling of Antibody
Antibody information: A therapeutic antibody (human IgG1 subtype)
Lot number: 5518.S9.011819
Figure 1: SEC HPLC analysis of purified SN38-ADC following the exact procedure of DCM11408 (Inset: UV/Vis spectra of SN38-ADC). Scale of the reaction: 3 mg of antibody.
Summary of the results:
R value (consider the total peaks) | 0.3 |
Average DAR based on R value | 3.3 |
Extent of antibody aggregation (%) | 13.6 |
Unreacted antibody (%) | 0 |
Unreacted SN38 (%) | 0 |
Recovery (%) | 73.6 |
Other Considerations
Drug-to-Antibody Ratio (DAR) and Characterization by UV and HPLC
In this kit, the target DAR is 3. To estimate the DAR, you can obtain the UV absorbance ratio (R) of your conjugate at 380 nm and 280 nm.
R = (A380)/(A280)
The unlabeled antibody has no absorbance at 380 nm. A SN38-ADC with DAR of 2-4 will have an R value of 0.19-0.36.
You can also use the following formula to calculate the estimated DAR (for reference only):
DAR = (34.43 x R)/(3.44 - R)
Dox: E280 nm = 6100 M-1cm-1 (data from CellMosaic) and E380 nm = 20985 M-1cm-1 (Nakatsuji M. et al. Human Lipocalin-Type Prostaglandin D Synthase-Based Drug Delivery System for Poorly Water-Soluble Anti-Cancer Drug SN-38. PLOS One 2015, 10(11): e0142206).
Antibody: E280 nm = 210,000 M-1cm-1 and no absorbance at 380 nm
Aggregation and Precipitation Issue for SN38 Labeling
SN38 is very hydrophobic. This kit is designed to minimize the aggregation and precipitation issues generally seen with SN38 labeling. Depending on the properties of your antibody, recovery will be 40-80%. If you are concerned with the aggregation, you can use size-exclusion chromatography (SEC) to check the extent of aggregation. If you do not have access to such a facility, you can send your sample to CellMosaic for analysis.
Recommended Storage Conditions
SN38 is linked to antibody through a releasable linker. Recommended use within 24 h and do not store SN38-ADC. If you need to store the ADCs, please dilute your ADC in Stabilization PBS buffer (5x). Aliquot and store the conjugate in a < -20°C freezer or lyophilize to dryness. You might still see some solid precipitate out during the storage using our stabilization buffer. Please centrifuge or filtrate before use. Avoid repeated freeze and thaw cycles. If the ADC is in a lyophilized powder, after dissolving, the solution should be used immediately within 24 h. The stability of your conjugate may be different due to your antibody and should be checked by SEC HPLC.
ADC Stabilizing Buffer
CellMosaic’s proprietary ADC stabilizing PBS buffer (5x) contains 5x PBS buffer and other stabilizers to prevent the hydrophobic drugs from interacting with each other during storage and causing the ADCs to precipitate out. Stabilization buffer also helps preserve the structure of the ADCs during lyophilization. The buffer is biocompatible and can be used directly for any in vitro and in vivo studies. For more information on the stabilization buffers, please check our website.
Submit Samples for HPLC Analysis
If you are submitting samples to CellMosaic for SEC analysis, please follow these instructions:
1) Dilute your un-conjugated antibody to 1 mg/mL in PBS buffer, then transfer 50 μL of the diluted solution to a 500 μL microcentrifuge tube. Label the vial properly.
2) Transfer 50 μL of ADC (non-diluted solution) to a 500 μL microcentrifuge tube and label the vial properly.
3) Ship your samples with a cold pack for overnight delivery.