|SDS||USER MANUAL CM11416
CellMosaic has designed this kit for labeling any F(ab')2 with monomethyl auristatin E (MMAE) using a valine-citruline p-aminobenzylcarbamate (VC-PAB) linker for early stage screening of your antibody.
This kit includes maleimide-activated VC-PAB-MMAE, which can be coupled directly to a thiol modified F(ab')2 through alkylation in a single step (similar method developed by Seattle Genetics: Sun et al. 2005, Bioconjugate Chem. 16, 1282-1290). The product is purified to remove any unreacted drug.
Kit is configured to conjugate 0.73 to 2.2 mg of one (CM11416x1) or three F(ab')2 (CM11416x3).
If you have less amount of F(ab')2 or other customization need, please select the customization radio button or fill out the information above.
- Name: Monomethyl auristatin E (MMAE) with Mal-Val-Cit-PAB linkage
- CAS number: 646502-53-6
- Mechanism of action: Inhibits cell division by blocking the polymerization of tubulin. VC-PAB linker is stable in extracellular fluid, but cleaved by cathepsin B once inside the tumor cell, thus activating the antimitotic mechanism
- Activities: Antioxidant, anti-inflammatory, anticancer, and insecticidal activities
Requirement for F(ab')2
- Preferably >90% pure by gel electrophoresis
- Total amount: 0.73-2.2 mg protein content measured by UV
Key Features of this PerKit™
- Offers a simple and easy way for site specific labeling of F(ab')2 with MMAE with minimum exposure to the toxin
- Cathepsin B cleavable VC-PAB (Ref. Doronina et. al. 2008, Bioconjugate Chem. 19, 1960-1963)
- Fast and easy preparation: 6 h preparation and <2 h hands-on time
- All reagents and supplies included for preparation and purification
- Easy to control the DAR if used together with the Thiol Assay Kit with Purification (Product#: CM90005)
- 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 loading.
Schematic diagram of the workflow for preparing F(ab')2-MMAE conjugates starting with 2.2 mg of IgG (volume of reagents varies if the amount of F(ab')2 is < 2.2 mg
F(ab’)2 information: F(ab’)2 was prepared using CellMosaic’s PerKit™ F(ab’)2 preparation kit (Cat#: CM51408) from a mouse IgG1 antibody.
Kit Lot number: 5525.S13.032919
Scale of the reaction: 0.5 mg
DAR of the final conjugates: 2 calculated based on the UV absorbance ratio (R) of the conjugate at 248 nm and 280 nm.
Figure 1. SEC HPLC analysis of F(ab')2 and purified F(ab')2-MMAE conjugates (UV at 220 nm)
Figure 2. HIC HPLC analysis of F(ab')2 and purified F(ab')2-MMAE conjugates (UV at 205 nm)
Thiol Assay Kit with Purification (Product #: CM90005): The reducing protocol is optimized for monoclonal IgG1 subtype to obtain an average 4 thiols per antibody (DAR = 4). For other IgG subtypes or polyclonal antibodies, the DAR may vary. For the best performance of the ADC and to obtain the desired DAR, you can purchase this kit separately and use it to perform an in-process thiol assay after the antibody reduction (Step C5 Note Section). The amount of reducing reagent can be adjusted based on the data to obtain your desired DAR.
Hydrophobic Interaction Chromatography (HIC) Buffer Set (Product#: CM02025): For ADCs prepared via a reduced thiol of the antibody, HIC HPLC is used to calculate the DAR and the heterogeneity of the ADCs. The conjugates are separated based on hydrophobicity. Antibodies loaded with the same number of drugs (same DAR) will have similar hydrophobicity and be eluted as a single peak. For a typical MMAE ADC, multiple peaks represent various amounts of drug-loaded ADCs. You will find examples of HIC HPLC profiles of MMAE ADCs of various antibodies in the Appendix. 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): SEC HPLC 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.