Antibody Methotrexate Conjugation Kit

Product Description

CellMosaic has designed this kit for antibody (IgG) labeling of methotrexate (formerly known as amethopterin) for early stage screening of your antibody.

Methotrexate is a chemotherapy agent and immune system suppressant on the World Health Organization's List of Essential Medicines, the most effective and safe medicines needed in a health system. This kit includes methotrexate N-hydroxysuccimide ester, which can be directly coupled to the antibody in a single step. The product is purified to remove any unreacted drug. 

This kit provides materials to conjugate 1 to 3 mg of antibody (IgG only) with methotrexate.

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.

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Drug Information

• Name: Methotrexate (amethopterin)
• CAS number: 59-05-2
• Mechanism of action: Competitively inhibits dihydrofolate reductase (DHFR), an enzyme that participates in tetrahydrofolate synthesis. Folic acid is essential for de novo synthesis of the nucleoside thymidine. In addition, folate is essential for biosynthesis of purine and pyrimidine bases. By inhibiting DHFR, methotrexate inhibits the synthesis of DNA, RNA, thymidylates, and proteins.
• Medical usage: Breast cancer, leukemia, lung cancer, lymphoma, and osteosarcoma

Key Features of this PerKit™

• Offers a simple and easy way to label IgG with methotrexate with minimum exposure to the chemotherapeutic drug
• Stable linkage
• Single conjugation step, fast and easy preparation: 6 h preparation with <1 h hands-on time
• All reagents and supplies included for preparation and purification
• DAR with average 3 to 5 methotrexate per antibody
• Included stabilizing buffer for long-term storage
• >99% conjugated products by size-exclusion chromatography (SEC) and free of any unreacted drugs
• Options to choose tailored services at CellMosaic after conjugation

After conjugation: you can choose to send your sample to CellMosaic for HPLC analysis.

Conjugation Chemistry

 Protocol

Example of methotrexate Labeling of Antibody

Antibody information: A therapeutic antibody (human IgG1 subtype)

Lot number: S283.S9.042320

Figure 1: SEC HPLC analysis of purified methotrexate-ADC (MTX-ADC) in PBS ADC stabilizing buffer. Two labeling reactions. Scale of the reaction: 0.5 mg of antibody each reaction. Not much aggregation was observed for high and low loading MTX-ADC.

Figure 2: Overlay of HIC HPLC analysis of unlabeled antibody, MTX-ADC with DAR of 1.65, and MTX-ADC with DAR of 3.84. 

Summary of the results:

Other Considerations

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

In this kit, the target DAR is 3-5. To estimate the DAR, you can obtain the UV absorbance ratio (R) of your conjugate at 302 nm and 280 nm.

 R = (A302)/(A280)

The unlabeled antibody will have an R value of 0.085. A MTX-ADC with DAR of 3-5 will have an R value of 0.31-0.41.

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

 DAR = (13.29 x R - 1.13)/(1.28 - R)

Methotrexate: E_{280 nm} = 17,224 M^-1cm^-1 (data from CellMosaic) and E_{302 nm} = 20268 M^-1cm^-1in PBS buffer according to data obtained from CellMosaic and based on the literature data (Touchette N.A.; Perry K. M.; Matthews, C. R. (1986) Folding of Dihydrofolate Reductase from Escherichia coli. Biochemistry 25, 5445-5462. Dawson, R. M. C., Elliot, D. C., Elliot, W. M., & Jones, K. M. (1969) in Data for Biochemical Research, p 206, Oxford University Press, Oxford. E_{302 nm} = 22,100 M^-1cm^-1 in 0.1N NaOH).

Antibody: E_{280 nm} = 210,000 M^-1cm^-1 and 17,904 M^-1cm^-1 at 302 nm (data obtained from CellMosaic)

Aggregation and Precipitation Issue 

Methotrexate is not considered very hydrophobic due to its two carboxylic acid groups. We have not seen any aggregations of the ADC caused by methotrexate labeling. However, we have observed lower recovery for ADC with higher loading compared to lower loading. 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

ADC labeled with methotrexate is relatively stable. Recommended use is within 2 weeks if you store methotrexate-ADC at 2-8°C. Based on our preliminary data, the conjugate made with this kit can remain stable in PBS buffer for a few days at 2-8°C (no long-term stability data). The stability of your conjugate may be different due to your antibody and should be checked by either HPLC or UV. If you need to store the ADCs for longer, please dilute your ADC in Stabilization PBS buffer (5x). Aliquot and store the conjugate in a freezer less than -20°C or lyophilize to dryness. Avoid repeated freeze and thaw cycles. 

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.