|SDS||USER MANUAL CM11413|
CellMosaic® has designed this personalized conjugation kit to work with any protein with a MW over 20 KDa. The customer supplies its own unmodified protein. Using the kit components, the customer converts their protein to a thiol-protein, followed by reaction of the maleimide-activated VC-PAB-MMAE to generate the protein-MMAE conjugates (a similar method developed by Seattle Genetics: Sun et al.2005,Bioconjugate Chem. 16, 1282-1290). One step purification typically provides the resulting protein-MMAE at greater than 95% purity.
This kit provides materials to conjugate 6.67-20 nmol of one (CM11413x1) or three protein samples (CM11413x3).
If you have less amount of protein or protein with MW below 20KDa, or other customization need, please select the customization radio button or fill out the information above.
Requirement for protein:
- Amount: 6.67-20 nmol
- MW: >20 KDa
- Preferably >90% pure by gel electrophoresis or SEC HPLC analysis
- Protein has some lysine residues
- 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
Key Features of this PerKit™
- Offers a simple and easy way to label protein 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
- 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 DOL.
Schematic diagram of the workflow for preparing protein-MMAE conjugates starting with 20 nmole of protein (volume of reagents varies if the amount of protein is < 20 nmole)
Example 1: MMAE Conjugation with BSA
Protein information: Bovine Serum Albumin (BSA), Kit Lot number: 5529.S14.072519
Scale of the reaction: 20 nmole following the exact protocol without any adjustment. Data provided for information only.
Specification of the final conjugates:
Calculated average DPR based on the A248 nm and A280 nm ratio: ~4
Calculated average DPR based on the MS data: ~3
% of unreacted antibodies: 0%; % of unreacted MMAE: 0%; Product yield: 65%
Figure 2: SEC/UV HPLC analysis of BSA (green trace) and purified conjugates (purple trace).
Figure 3: MS analysis of BSA (left) and purified conjugates (right) (data acquired by MDS SCIEX 4800 MALDI TOF Analyzer).
Example 2: MMAE Conjugation with Fab
Fab information: Fab was prepared following a similar protocol as CellMosaic’s PerKit™ Fab preparation kit (Cat#: CM51407) from a rabbit IgG antibody.
Kit Lot number: 5529.S14.072519
Scale of the reaction: 2 nmole (limited material available) following a modified protocol. Data provided for information only.
Figure 4: HIC HPLC analysis of Fab and purified conjugates
Figure 5: SEC HPLC analysis of Fab and purified conjugates
Hydrophobic Interaction Chromatography (HIC) Buffer Set (Product#: CM02025): For PDCs prepared via surface amines of the protein, hydrophobic interaction chromatography (HIC) HPLC can be used to check if a protein is labeled or not. However, due to the highly heterogeneous nature of surface amine labeling, protein loaded with the same number of drugs (same DPR) may have slightly different hydrophobicity. For a typical MMAE PDC, 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): VC-PAB-MMAE is very hydrophobic. This kit is designed to minimize the aggregation and precipitation issues generally seen with MMAE labeling. However, you may still notice some solid precipitate out or PDC aggregation during the reaction. The precipitate will be removed during purification. Depending on the properties of your protein, 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. 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 protein and the PDC, you can estimate how much aggregation is in the PDC. 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): 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, which would cause the conjugates to precipitate out. Stabilization buffer also helps preserve the structure of the PDC 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.