Antibody MMAF Conjugation Kit (with VC-PAB Linker)

Product Description

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 with a charged C-terminal phenylalanine residue. The kit includes maleimide-activated MMAF (Mc-VC-PAB-MMAF), which can be coupled directly to the antibody after reduction through alkylation in a single step (a method developed by Seattle Genetics: Sun et al. 2005, Bioconjugate Chem. 16, 1282-1290). The product is purified to remove any unreacted drugs.

This kit provides materials to conjugate 1-3 mg of one (CM11425) or three (CM11425x3) antibody samples (IgG only).

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: Heat sensitive and ship with cold packs, choose overnight (ships Mon.-Thur.) or 2-day delivery (ships Mon.-Wed.) at checkout.

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

1. Preferably >90% pure by gel electrophoresis
2. Total amount: 1-3 mg protein content measured by UV

Key Features of this PerKit™

• Offers a simple and easy way to label MMAF with minimum exposure to the chemotherapeutic dru
• Cathepsin B cleavable VC-PAB linkage (Ref. Doronina et. al. 2008, Bioconjugate Chem. 19, 1960-1963)
• Fast and easy preparation: 6 h preparation with <2 h hands-on time
• All reagents and supplies included for preparation and purification
• Minimum aggregation with average 2-4 drugs per antibody and free of any unreacted drugs
• Easy to control the DAR if used together with the Thiol Assay Kit with Purification (Product#: CM90005)
• Included stabilizing buffer for long-term storage
• Options to choose tailored services at CellMosaic after conjugation

After conjugation: you can choose to send your sample to CellMosaic for HPLC analysis and calculation of the DAR.

Conjugation Chemistry

Schematic diagram of the workflow for preparing antibody-MMAF conjugates starting with 3 mg of IgG (volume of reagents varies if the amount of IgG is < 3 mg)

Example of Mc-VC-PAB-MMAF Labeling of Antibody

Antibody information: A therapeutic antibody (human IgG1 subtype)

Lot number: 5524.S13.021119

Scale of the reaction: 1 mg of antibody (protein content)

Figure 1: SEC HPLC analysis of purified MMAF-ADC with VC-PAB linker.	Figure 2. HIC HPLC analysis of antibody (red trace) and purified conjugates (blue trace)

Summary of the results:

Other Considerations

Drug-to-Antibody Ratio (DAR) and Characterization by UV and HPLC

In this kit, the target DAR is 3-4. Depending on your antibody, you may achieve a lower DAR.

To estimate the DAR, you can obtain the UV absorbance ratio (R) of your conjugate at 248 nm and 280 nm. 

 R = (A248)/(A280)

The unlabeled antibody will have an R value of 0.4 – 0.5. An MMAF-ADC with DAR of 3 – 5 will have an R value of 0.65 – 0.80.

You can also use the following formula to calculate the estimated DAR (only for reference):

 DAR = (21 x R - 9)/(1.615 - 0.1425 x R)

Note: The UV absorbance of the MMAF in an ADC can vary greatly depending on many factors, such as aggregation and stacking. Therefore, the R value for an ADC can differ greatly for different antibodies and should be determined experimentally. The calculation for the DAR using this formula is only for reference only.

For more accurate DAR calculation and to check the homogeneity of the ADC, you can analyze it by hydrophobic interaction chromatography (HIC). If you do not have access to such a facility setup, you can send your sample to CellMosaic for analysis.

Aggregation and Precipitation Issue for MMAF Labeling

Mc-VC-PAB-MMAF is very hydrophobic. This kit is designed to minimize the aggregation and precipitation issues generally seen with MMAF 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 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

Recommend storage at 2-8°C for short-term. Do not freeze in PBS buffer. Based on our preliminary data, the conjugate made with this kit can remain stable in PBS buffer at 2-8 °C for few days. You may notice the increase amount of the aggregation after storing for sometimes. The stability of your conjugate may be different due to your antibody and should be checked either by HPLC or UV.

If you need to store the ADCs for long term, please dilute your ADC in Stabilization PBS buffer (5x). Aliquot and store the conjugate in a < -20°C freezer or lyophilize to dryness. Avoid repeated freeze and thaw cycles. If the ADC is in a lyophilized powder, after dissolving, the solution should be used within few days.

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 and HIC 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.