(M1030-01-01) Comparison of Linker-Payload Cleavage Methods of the Antibody Drug Conjugate Anetumab-MMAE to Determine the Conjugated Payload MMAE Concentration by LC-MS/MS

Purpose: Determination of the conjugated payload concentration from an antibody drug conjugate can be obtained with several different methods. The purpose of this poster is to report the pros and cons on three different mechanisms of cleaving the payload from the ADC Anetumab-MMAE prior to analysis by LC-MS/MS. The three mechanisms tested will be a simple pellet digestion, Protein A capture/elution/digestion and Anti Human IgG1 Fc capture/elution/digestion using papain as the digestion enzyme.

Methods: Anetumab-MMAE is an antibody drug conjugate (ADC) composed of a human anti-mesothelin antibody linked to the anti-mitotic cytotoxic agent monomethyl auristatin (MMAE). Anetumab-MMAE was spiked into rat plasma and 25 μL of sample aliquotted. For the pellet digestion method MTBE was added, mixed, centrifuged and the supernatant was transferred to remove the free MMAE. The remaining plasma aliquot was then evaporated to remove the MTBE. In order to measure the conjugated MMAE papain was added to cleave the MMAE-antibody linker and extracted with MTBE. The Protein A capture method utilized Peirce Protein A spin plates. The 96 well plates were conditioned with binding buffer and the plasma aliquot was placed on the device, incubated, rinsed with binding buffer and eluted using elution buffer. Papain was then added to cleave the MMAE from the linker. MTBE was then added to extract the free MMAE for analysis. The magnetic bead capture method begins with conditioning the streptavidin coated beads. Once conditioned the beads are mixed with a solution of biotinylated Anti Human Fc specific antibodies to form a link. The diluted plasma aliquot is added to the mixture, incubated to form the bond between the capture antibody and Anetumab and eluted.  Papain is added to digest the MMAE from the linker and is extracted with MTBE. In order to achieve the lowest limit of quantitation (LLOQ) for MMAE a SCIEX 7500+ triple quadrupole mass spectrometer operating in positive ion mode was used. The HPLC system were Shimadzu 40 AD pumps operating with a binary gradient at a flowrate of 0.700 mL/min. Separation was achieved using a Synergi Hydro-RP column (100 X 3 mm).

Results: The pellet digestion is the simplest method of the three and only one sample aliquot is required to determine the MMAE free payload and conjugated payload. This low sample aliquot volume is most useful in mouse studies where sample volume is limited. This method can also have consequences of selectivity problems in the LC-MS/MS analysis since the entire pellet is being digested and analyzed. the Protein A Capture method is simpler than a bead capture method since the protein A is already in a 96 well format. The cost per sample is $4 which is considerably more than the cost of a pellet digestion (pennies per well) but offers better selectivity since it will only capture the Fc region of IgG antibodies. Since it is not species specific it will also capture the IgG antibodies from other species and that may cause a problem with interfering non-human compounds after digestion. The most selective, labor intensive and expensive method is a magnetic bead capture using the Anti Human IgG1 Fc antibody. The labor required to prep the beads, bind the biotin to the streptavidin, capture the ADC, wash and elute is intensive compared to the other two methods. The data generated from the bead capture method will be the most selective since only Human IgG1 antibodies will be captured and digested. This offers an option for analysis if the other two methods are not selective enough. The data suggests that each method is accurate and precise and can be used to quantify MMAE conjugated to a cleavable linker.

Conclusion: Each method tested is accurate and precise enough to be used in a regulated environment for the ADC Anetumab-MMAE . If selectivity problems are encountered a Protein A and Anti Human IgG1 capture may be necessary.