Product Description
| Download Documents | |
| SDS | USER MANUAL CM11410 (1-3 mg scale) |
| USER MANUAL CM11410.01 (0.1 mg scale) | |
CellMosaic has designed this kit for antibody (IgG type) labeling of mertansine (DM1) with SMCC (succinimidyl trans-4-(maleimidylmethyl)cyclohexane-1-carboxylate) linker for early stage screening of your antibody.
This kit includes SMCC and DM1, which can be coupled to the antibody sequentially via surface amines (a method developed by Immunogen). The product is purified to remove any unreacted drug.
This kit provides materials to conjugate 1 to 3 mg of one (CM11410) or three (CM11410x3) antibody samples (IgG), and 0.1 mg of one (CM11410.01) or three (CM11410.01x3) antibody samples.
If you have 0.1 to 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.
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Drug Information
- Name: Mertansine (DM1)
- CAS number: 139504-50-0
- Mechanism of action: Inhibits cell division by blocking the polymerisation of tubulin
Requirement for Antibody
- Preferably >90% pure by gel electrophoresis
- Total amount: 1-3 mg or 0.1 mg of protein content as measured by UV.
Key Features of this PerKit™
- Offers a simple and easy way to label IgG with DM1 with minimum exposure to the toxin
- Stable linkage
- Fast and easy preparation: 6 h preparation and <2 h hands-on time
- All reagents and supplies included for preparation and purification
- Average 2-4 DM1 labeling per antibody
- Less than 5% of antibody aggregation and >99% of conjugated products by SEC (size-exclusion chromatography)
- Options to choose tailored services at CellMosaic after conjugation
After conjugation: you can choose to send your sample to CellMosaic for HPLC analysis and determination of the DAR.
Conjugation Chemistry
Schematic diagram of the workflow for preparing antibody-DM1 conjugates starting with 3 mg of IgG (volume of reagents varies if the amount of IgG is < 3 mg)
Typical Kit Performance Data (LC Analysis, CellMosaic)
Antibody information: A therapeutic antibody (human IgG1 subtype), Kit Lot number: 5513.S9.053018
Figure 1: SEC HPLC analysis of antibody (blue color) and purified ADC-DM1 (red color) (Inset: UV/Vis spectra of the unlabeled antibody and ADC-DM1.) Scale of the reaction: 1 mg (CM11410.1)
Summary of the result:
|
Test Reactions |
1 mg Scale Reaction (CM11410.1) |
0.1 mg Scale Reaction (CM11410.01) |
|
R value (only consider the 10.46 min peak product) |
0.78 |
0.72 |
|
Average DAR based on R value |
3.24 |
2.7 |
|
Maleimide groups per antibody after SMCC labeling |
3.8 |
3.2 |
|
Extend of antibody aggregation (%) |
3.8 |
3.16 |
|
Unreacted antibody (%) |
0 |
0 |
|
Unreacted DM1 (%) |
0 |
0 |
|
Recovery (%) |
38 |
46 |
R value (A252 nm/A280 nm) of unlabeled antibody: 0.515
The higher the average DAR of ADC-DM1, the greater the percentage of the antibody aggregates and the lower the overall recovery.
Related Products
Maleimide Assay Kit (Cat. CM90002): The average maleimide groups per antibody is 4 after SMCC labeling. If necessary, you can purchase this kit separately from CellMosaic to assay the maleimide content. The kit is simple and easy to use. Approximate total assay time is 1 h.
Hydrophobic Interaction Chromatography (HIC) Buffer Set (Product#: CM02025): For ADCs prepared via surface amines of the antibody, hydrophobic interaction chromatography (HIC) HPLC can be used to check if an antibody is labeled or not. However, due to the highly heterogeneous nature of surface amine labeling, antibody loaded with the same number of drugs (same DAR) may have slightly different hydrophobicity. For a typical DM1 ADC, a broad peak will be seen without clear separation of the peaks. CellMosaic offers HIC buffer for our customers to use with any HIC column. The CM02025 product sheet contains all of the information and methodology needed to run an HIC HPLC analysis. If you do not have access to an HPLC facility, you can send your sample to CellMosaic for analysis.
Size Exclusion Chromatography (SEC) HPLC Protein Standards (Product #: CM92004 and CM92005): DM1 is very hydrophobic. This kit is designed to minimize the aggregation and precipitation issues generally seen with DM1 labeling. However, you may still notice some solid precipitate out or ADC aggregation during the reaction. The precipitate will be removed during purification. Depending on the properties of your antibody, recovery will be 30-60%. If you are concerned with the aggregation, you can use size exclusion chromatography (SEC) to check the extent of aggregation. SEC separates the conjugates by apparent molecular weight (MW) or size in aqueous solution. The larger the MW of the conjugate, the earlier it elutes. By comparing the SEC profile of unlabeled IgG and the ADC, you can estimate how much aggregation is in the ADC. CellMosaic offers two SEC standards for our customers to use with any SEC column. The CM92004 product sheet contains all of the information and methodology you need to run an SEC HPLC analysis. If you do not have access to an HPLC facility, you can send your sample to CellMosaic for analysis.
ADC stabilizing PBS buffer (5x) (Product #: CM02022): CM02022 contains 5x PBS buffer and other stabilizers to prevent the hydrophobic drugs from interacting with each other during storage, which would cause 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
