Product Description
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CellMosaic® has designed this kit for antibody (IgG) labeling of monomethyl auristatin F (MMAF) with valine-citruline p-aminobenzylcarbamate (VC-PAB) (a releasable linkage). MMAF is a new antimitotic auristatin derivative featuring a charged C-terminal phenylalanine residue. The kit includes maleimide-activated MMAF (Mc-VC-PAB-MMAF), which can be directly coupled to the antibody following reduction and alkylation in a single step (a method developed by Seattle Genetics: Sun et al., 2005, Bioconjugate Chem. 16, 1282-1290). The product is then purified to remove any unreacted drugs.
This kit provides materials to conjugate 1-3 mg of one (CM11425x1) or three (CM11425x3) antibody samples (IgG only).
If you have a smaller amount of IgG, a non-IgG type antibody, or other customization needs, please select the customization option and/or fill out the information above.
Drug Information
- Name: Monomethyl auristatin F (MMAF) with Mc-VC-PAB linker
- CAS number: N/A
- 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, activating the antimitotic mechanism.
- Activities: Antioxidant, anti-inflammatory, anticancer, and insecticidal activities.
- Selected References: 1) Doronina SO, Mendelsohn BA, Bovee TD, et al. Enhanced activity of monomethylauristatin F through monoclonal antibody delivery: effects of linker technology on efficacy and toxicity. Bioconjug Chem 2006;17: 114–24. 2) Polson AG, et al. Antibody-drug conjugates for the treatment of non-Hodgkin's lymphoma: target and linker-drug selection. Cancer Res. 2009;69(6):2358−2364.
Requirement for Antibody
- Preferably >90% pure by gel electrophoresis
- Total amount: 1-3 mg protein content as measured by UV
Key Features of this PerKit™
- Simple and efficient labeling of IgG with MMAF, minimizing toxin exposure .
- Features Cathepsin B cleavable VC-PAB linker (Ref. Doronina et al., 2008, Bioconjugate Chem. 19, 1960-1963).
- Delivers an average of 4 MMAF molecules per antibody.
- Fast preparation: 4 hours total, with less than 1 hour of hands-on time.
- Includes all necessary reagents and supplies for preparation and purification.
- Achieves over 95% conjugation, free from unreacted MMAE.
- Post-conjugation services available at CellMosaic for HPLC analysis and calculation of the DAR.
Conjugation Chemistry
Protocol
Schematic workflow diagram for preparing antibody-MMAF conjugates with VC-PAB linkage, starting with 3 mg of IgG. (Reagent volume will vary if the amount of IgG is less than 3 mg).
Other Considerations
Drug-to-Antibody Ratio (DAR) and Characterization by UV
In this kit, the target DAR is 4. Depending on your antibody, you may achieve a slightly higher or lower DAR.
To estimate the DAR, you can calculate the UV absorbance ratio (R) of your conjugate at 248 nm and 280 nm using the following formula.
R = (A248)/(A280)
Unlabeled antibody typically has an R value between 0.4 and 0.5.
MMAE-ADC with a DAR of 3 to 5 have an R value between 0.65 – 0.80.
You can also use the following formula to estimate the DAR (for reference only):
DAR = (21 x R - 9)/(1.615 - 0.1425 x R)
Note: The UV contribution of the VC-PAB-MMAF to the ADC is experimentally determined at CellMosaic. The UV absorbance of the VC-PAB-MMAF in an ADC can vary significantly due to factors like aggregation and stacking. Therefore, the R value for an ADC may differ greatly depending on the antibodies and should be determined experimentally. The DAR calculation using this formula is for reference purpose only.
Aggregation and Precipitation Issue for MMAF Labeling
Mc-MMAF with VC-PAB is highly hydrophobic. This kit is designed to minimize the aggregation and precipitation issues typically encountered with Mc-VC-PAB-MMAF labeling. However, you may still notice some solid precipitate or ADC aggregation during the reaction. The precipitate will be removed during purification. Depending on the properties of your antibody, recovery may range from 40-80%.
If you are concerned about aggregation, you can use size exclusion chromatography (SEC) to assess the extent of aggregation.
If you do not have access to an HPLC facility, you can send your sample to CellMosaic for analysis.
Recommended Storage Conditions
Recommend storing at 2-8°C for short-term. Do not freeze in PBS buffer. Based on our preliminary data, the conjugate made with this kit remains stable in PBS buffer at 2-8 °C for several weeks. However, you may notice an increased amount of aggregation after some time. The stability of your conjugate may vary depending on your antibody and should be checked either by HPLC or UV.
For long-term storage, dilute your ADC in Stabilization PBS Buffer (5x) (included in this kit). Aliquot and store the conjugate in a freezer at temperatures below -20°C or lyophilize to dryness. Avoid repeated freeze-thaw cycles. If the ADC is in lyophilized powder form, once dissolved, the solution should be used within a few days.
ADC Stabilizing Buffer
CellMosaic’s proprietary ADC stabilizing PBS buffer (5x) contains 5x PBS buffer and other stabilizers designed to prevent hydrophobic drugs from interacting with each other during storage, which can lead to ADCs precipitation. The Stabilizing Buffer also helps preserve the structure of the ADCs during lyophilization. This biocompatible buffer can be used directly for both in vitro and in vivo studies. For more information on stabilization buffers, please visit our website.
Submit Samples for HPLC Analysis
If you are submitting samples to CellMosaic for SEC and HIC analysis, please follow these instructions:
- Visit CellMosaic’s HPLC analysis page, select SEC HPLC Analysis (Product# AS0023) and HIC HPLC Analysis (Product#: AS0025), choose the quantity (number of samples. Bulk discounts available for multiple samples), and submit your order. Alternatively, you can email info@cellmosaic.com for a quote and to place the order.
- Dilute your un-conjugated antibody in PBS buffer to a concentration of 1 mg/mL. Transfer 50 μL of the diluted solution into a 500 μL micro-centrifuge tube and label the vial properly.
- Transfer 50 μL of ADC (non-diluted solution) into a 500 μL micro-centrifuge tube and label the vial properly.
- Ship your samples with a cold pack for overnight delivery.
Related Products
ADC Control hIgG1-MMAF with VC-PAB-Linker (Product #: CM51101): A control ADC made with a non-binding human IgG1 subtype. This product contains an average of 4 MMAF molecules per antibody and has low endotoxin levels, making it suitable for in vitro and in vivo studies.
HRP-VC-PAB-MMAF (1:1) Conjugate (Product #: CM53217): An assay reagent used for HRP based assay.
