Native-MS analysis of monoclonal antibody conjugates by Fourier transform ion cyclotron resonance mass spectrometry

Antibody drug conjugates (ADCs) are an important class of therapeutic molecule currently being used to treat HER2-positive metastatic breast cancer, relapsed or refractory Hodgkin lymphoma, systemic anaplastic large cell lymphoma, relapsed or refractory B-cell precursor acute lymphoblastic leukemia and acute myeloid leukemia. An ADC typically consists of a small molecule or peptide-based cytotoxic moiety covalently linked, via lysine or cysteine residues, to amonoclonal antibody (mAb) scaffold. Mass spectrometric (MS) characterization of these molecules afford highly accurate molecular weight (MW) and drug-to-antibody ratio (DAR) determination, and is typically performed using orthogonal acceleration time-of-flight (oa-ToF) analysers and more recently Orbitrap instruments. Herein we describe for the first time the use of a 15 Tesla solariX Fourier transform ion cyclotron mass spectrometer to characterize an IgG1 mAb molecule conjugated with biotin via native lysine and cysteine residues, under native-MS and solution conditions. The cysteine biotin conjugates remained fully intact, demonstrating the ability of the FT-ICR to maintain the noncovalent interactions and efficiently transmit labile protein complexes. Native-MS was acquired and is displayed in magnitude mode using a symmetric Hann apodisation function. Baseline separation is achieved on all covalent biotin additions, for each charge state, for both the lysine and cysteine biotin-conjugates. Average DAR values obtained by native-MS for the lysine conjugate are compared to those derived by denaturing reversed phase liquid chromatography using an oa-ToF MS system (1.56 ±0.02 versus 2.24 ±0.02 for a 5-molar equivalent and 3.99 ±0.09 versus 4.43 ±0.01 for a 10-molar equivalent, respectively). Increased DAR value accuracy can be obtained for the higher biotin load, when using standard ESI conditions as opposed to nanoESI native-MS conditions. Both denatured LC-MS and native-MS spectral data were deconvoluted using a parsimonious based algorithm, without the need for parameter adjustment

Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting

The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody-capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics

Synthetically Modified Viral Capsids as Versatile Carriers for Use in Antibody-Based Cell Targeting

The present study describes an efficient and reliable method for the preparation of MS2 viral capsids that are synthetically modified with antibodies using a rapid oxidative coupling strategy. The overall protocol delivers conjugates in high yields and recoveries, requires a minimal excess of antibody to achieve modification of more than 95% of capsids, and can be completed in a short period of time. Antibody–capsid conjugates targeting extracellular receptors on human breast cancer cell lines were prepared and characterized. Notably, conjugation to the capsid did not significantly perturb the binding of the antibodies, as indicated by binding affinities similar to those obtained for the parent antibodies. An array of conjugates was synthesized with various reporters on the interior surface of the capsids to be used in cell studies, including fluorescence-based flow cytometry, confocal microscopy, and mass cytometry. The results of these studies lay the foundation for further exploration of these constructs in the context of clinically relevant applications, including drug delivery and in vivo diagnostics